Production of Polyunsaturated Fatty Acids, Novel Biosynthesis Genes, And Novel Plant Expression Constructs

ABSTRACT

The present invention relates to a method for the production of unsaturated fatty acids with at least two double bonds. The invention furthermore relates to the use of nucleic acid sequences SEQ ID NO: 1, 3, 5, 9, and 11 encoding polypeptides having desaturase or elongase activity in the method and for generating a transgenic organism, preferably a transgenic plant or a transgenic microorganism, with an increased content of fatty acids, oils or lipids with unsaturated C 18 -, C 20 -, or C 22 -fatty acids, and to their homologs or derivatives, to gene constructs encompassing these genes, and to their use alone or in combination with biosynthesis genes of polyunsaturated fatty acids. The invention also relates to multiexpression cassettes for seed-specific expression, and to vectors or organisms which encompass a desaturase gene alone or in combination with further desaturases and/or elongase genes or homologs using said expression cassettes.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/565,799 filed Sep. 24, 2009, which is a divisional application ofU.S. application Ser. No. 10/250,553 filed Jul. 2, 2003, which is anational stage application (under 35 U.S.C. 371) of PCT/EP2002/00462filed Jan. 18, 2002, which claims benefit of Germany application10102337.2 filed Jan. 19, 2001. The entire content of eachabove-mentioned application is hereby incorporated by reference in itsentirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_List_(—)12810_(—)00928DIV. The size ofthe text file is 469 KB, and the text file was created on Jan. 2, 2012.

FIELD OF THE INVENTION

The present invention relates to a method for the production ofunsaturated fatty acids with at least two double bonds and/or a methodfor the production of triglycerides with an increased content ofpolyunsaturated fatty acids with at least two double bonds. Theinvention furthermore relates to the advantageous use of the nucleicacid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or 11 in themethod and for generating a transgenic organism, preferably a transgenicplant or a transgenic microorganism, with an increased content of fattyacids, oils or lipids with unsaturated C₁₈-, C₂₀-, or C₂₂-fatty acids.

The invention furthermore relates to novel desaturases with the [lacuna]in the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:11 or its homologs, derivatives and analogs, and to gene constructsencompassing these genes or their homologs, derivatives or analogs, andto their use alone or in combination with biosynthesis genes ofpolyunsaturated fatty acids as shown advantageously in SEQ ID NO: 7 andSEQ ID NO: 9.

In addition, the invention relates to isolated nucleic acid sequences;expression cassettes comprising the nucleic acid sequences, vectors andtransgenic organisms comprising at least one nucleic acid sequence orone expression cassette. In addition, the invention relates tounsaturated fatty acids with at least two double bonds and totriglycerides with an increased content of unsaturated fatty acids withat least two double bonds, and to their use.

Moreover, the invention relates to multiexpression cassettes forseed-specific expression, and to vectors or organisms which encompass adesaturase gene alone or in combination with further desaturases withthe sequence SEQ ID NO:7 and/or elongase genes with the sequence SEQ IDNO: 9 or its homologs, derivatives or analogs using said expressioncassettes.

BACKGROUND OF THE INVENTION

A series of products and by-products of naturally occurring metabolicprocesses in microorganisms, animal cells and plant cells has utilityfor a wide array of industries, including the feed, food, cosmetics andpharmaceutical industries. These molecules, which are collectivelytermed “fine chemicals”, also include, for example, lipids and fattyacids, one representative class of which are the polyunsaturated fattyacids. Fatty acids and triglycerides have a multiplicity of uses in thefood industry, in animal nutrition, in cosmetics and in thepharmacological sector. Depending on whether they take the form of freesaturated or unsaturated fatty acids or else triglycerides with anincreased content of saturated or unsaturated fatty acids, they aresuitable for a variety of uses; thus, for example, polyunsaturated fattyacids (PUFAs) are added to baby formula for increasing the nutritionalvalue. Moreover, PUFAs have a positive effect on the cholesterol levelin the blood of humans and are therefore useful for protection againstheart disease. Thus, they are used in a variety of dietetic foods or inmedicaments.

Microorganisms which are particularly useful for the production of PUFAsare microorganisms such as Thraustochytria or Schizochytria strains,algae such as Phaeodactylum tricornutum or Crypthecodinium species,ciliates such as Stylonychia or Colpidium, fungi such as Mortierella,Entomophthora or Mucor. Through strain selection, a number of mutantstrains of the respective microorganisms have been developed whichproduce an array of desirable compounds including PUFAs. However, theselection of strains in which the production of a particular molecule isimproved is a time-consuming and difficult process.

Alternatively, fine chemicals can conveniently be produced viaproducing, on a large scale, plants which have been developed in such away that they produce the abovementioned PUFAs. Particularly well suitedplants for this purpose are oil crop plants which comprise large amountsof lipid compounds, such as oilseed rape, canola, linseed, soybean,sunflower, borage and evening primrose. However, other useful plantscomprising oils or lipids and fatty acids are also well suited asmentioned in the detailed description of this invention. By means ofconventional breeding, an array of mutant plants has been developedwhich produce a spectrum of desirable lipids and fatty acids, cofactorsand enzymes. However, the selection of novel plant cultivars in whichthe production of a particular molecule is improved is a time-consumingand difficult process or indeed impossible if the compound does notoccur naturally in the respective plant, as is the case ofpolyunsaturated C₁₈-, C₂₀-fatty acids and C₂₂-fatty acids and those withlonger carbon chains.

Owing to the positive properties of unsaturated fatty acids, there hasbeen no lack of attempts in the past to make available genes which areinvolved in the synthesis of fatty acids or triglycerides for theproduction, in various organisms, of oils with a modified content ofunsaturated fatty acids. Thus, WO 91/13972 and its US equivalentdescribe a Δ9-desaturase. A Δ15-desaturase is claimed in WO 93/11245 anda Δ12-desaturase is claimed in WO 94/11516. Δ6-desaturases are describedin WO 93/06712, U.S. Pat. No. 5,614,393, WO 96/21022 and WO 99/27111.Further desaturases are described, for example, in EP-A-0 550 162, WO94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukeyet al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. A Δ6-palmitoylACP desaturase is described and claimed in WO 96/13591. However, thebiochemical characterization of the various desaturases is incomplete asyet since the enzymes, being membrane-bound proteins, can only beisolated and characterized with great difficulty (McKeon et al., Methodsin Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem.,26, 1988: 777-792).

In yeasts, both a shift in the fatty acid spectrum toward unsaturatedfatty acids and an increase in productivity have been found (see Huanget al., Lipids 34, 1999: 649-659, Napier et al., Biochem. J., Vol. 330,1998: 611-614). However, the expression of the various desaturases intransgenic plants did not show the desired success. While a shift in thefatty acid spectrum toward unsaturated fatty acids was demonstrated, itemerged that the synthesis performance of the transgenic plants droppeddrastically, i.e. only smaller amounts of oils were isolated comparedwith the original plants.

Neither yeasts nor plants naturally produce polyunsaturated C₂₀- and/orC₂₂-fatty acids with at least two double bonds in the fatty acidmolecule, such as arachidonic acid (ARA) and/or eicosapentaenoic acid(EPA) and/or docosahexaenoic acid (DHA).

This is why there still exists a great demand for novel genes whichencode enzymes which are involved in the biosynthesis of unsaturatedfatty acids and which make possible their production on an industrialscale. None of the prior-art biotechnological methods for the productionof polyunsaturated fatty acids yields the abovementioned fatty acids ineconomically useful quantities.

It is an object of the present invention to provide further enzymes forthe synthesis of polyunsaturated fatty acids and to use these enzymes,with or without other enzymes, in a method for the production ofpolyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the polypeptide alignment of the coding region of Pp_des 6(top row; SEQ ID NO: 8) with the EST sequence of PT001078032R (bottomrow; SEQ ID NO: 2).

FIG. 2 shows the polypeptide alignment of coding regions of Ma_des12(top sequence; Accession Number AF110509) with PT00107001OR (SEQ ID NO:6).

FIG. 2 a shows the polypeptide alignment of coding regions of Ma_des12(top sequence; Accession Number AF110509) with PT001072031R (SEQ ID NO:12).

FIG. 3 shows the polypeptide alignment of coding regions of a PCRproduct of the primer pair F6a and R4a2 encoding a Phaeodactylumdesaturase fragment (top row; SEQ ID NO: 19) with the Streptomycescoelicolor sequence T36617 (bottom row).

FIG. 4 shows the polypeptide alignment of coding regions of Pp_des6 (toprow; SEQ ID NO: 8) in comparison with Pt_des6 (bottom row; SEQ ID NO:4).

FIG. 5 shows the polypeptide alignment of coding regions of Pp_des6 (toprow; SEQ ID NO: 8) in comparison with Pt_des5 (bottom row; SEQ ID NO:2).

FIG. 6 shows the polypeptide alignment of coding regions of theMortierella alpina D 12-desaturase, Ma_des12 (top row; Accession NumberAF110509) with the homologous Phaeodactylum tricornutum sequence,Pt_des12 (bottom row; SEQ ID NO: 6).

FIG. 7 shows the polypeptide alignment of coding regions of theMortierella alpina D 12-desaturase, Ma_des12, (top row; Accession NumberAF110509) with the homologous sequence of Phaeodactylum tricornutumclone PT001072031R, Pt_des12.2 (bottom row; SEQ ID NO: 12).

DETAILED DESCRIPTION OF THE INVENTION

We have found that this object is achieved by the novel method for theproduction of fatty acid esters with an increased content ofpolyunsaturated fatty acids with at least two double bonds, whichcomprises introducing, into a fatty-acid-ester-producing organism, atleast one nucleic acid sequence selected from the group consisting of

-   -   a) a nucleic acid sequence with the sequence shown in SEQ ID NO:        1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,    -   b) nucleic acid sequences which, owing to the degeneracy of the        genetic code, are obtained by backtranslating the amino acid        sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or        SEQ ID NO: 12,    -   c) derivatives of the nucleic acid sequence shown in SEQ ID NO:        1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encode        polypeptides with the amino acid sequences shown in SEQ ID NO:        2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least        50% homology at the amino acid level, without essentially        reducing the enzymatic action of the polypeptides,        growing the organism, and isolating the fatty acid esters        present in the organism.

The nucleic acid sequences used in the method according to the inventionare isolated nucleic acid sequences which encode polypeptides with Δ5-,Δ6- or Δ12-desaturase activity.

It is advantageous to produce fatty acid esters with polyunsaturatedC₁₈-, C₂₀- and/or C₂₂-fatty acid molecules with at least two doublebonds in the fatty acid ester by the method according to the invention.These fatty acid molecules preferably comprise three, four or fivedouble bonds and advantageously lead to the synthesis of arachidonicacid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

The fatty acid esters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fattyacid molecules can be isolated from the organisms used for theproduction of the fatty acid esters in the form of an oil or lipid forexample in the form of compounds such as sphingolipids,phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerides, diacyl glycerides, triacyl glycerides or other fatty acidesters comprising the polyunsaturated fatty acids with at least twodouble bonds.

Suitable organisms for the production by the method according to theinvention are, in principle, all prokaryotic or eukaryotic organismssuch as prokaryotic or eukaryotic microorganisms such as Gram-positiveor Gram-negative bacteria, fungi, yeasts, algae, ciliates, animal orplant cells, animals or plants such as mosses, dicotyledonous ormonocotyledonous plants. It is advantageous to use, in the methodaccording to the invention, organisms which belong to the oil-producingorganisms, that is to say which are used for the production of oils,such as microorganisms such as Crypthecodinium, Thraustochytrium,Phaeodactylum and Mortierella, Entomophthora, Mucor, and other algae orfungi, and animals or plants, in particular plants, preferably oil cropplants which contain large amounts of lipid compounds, such as soybean,peanut, oilseed rape, canola, sunflower, safflower, evening primrose,linseed, borage, trees (oil palm, coconut) or crops such as maize,wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper,Tagetes, solanaceous plants such as potato, tobacco, aubergine andtomato, Vicia species, pea, alfalfa or bush plants (coffee, cacao, tea),Salix species, and also perennial grasses and fodder crops. Plantsaccording to the invention which are especially preferred are oil cropplants such as soybean, peanut, oilseed rape, canola, linseed, eveningprimrose, sunflower, safflower or trees (oil palm, coconut).

The method according to the invention comprises either breeding asuitable transgenic organism or transgenic microorganism or breedingtransgenic plant cells, tissues, organs or intact plants comprising thenucleotide sequences according to the invention of SEQ ID NO: 1, 3, 5 or11, if appropriate in connection with the sequences shown in SEQ ID NO:7 and/or SEQ ID NO: 9 alone or in combination with sequences ofexpression constructs from SEQ ID NO: 13-17 or their homologs,derivatives or analogs or a gene construct which encompasses SEQ ID NO:1, 3, 5 or 11, if appropriate in connection with SEQ ID NO: 7 and/or 9or their homologs, derivatives or analogs, or a vector comprising thissequence or the gene construct which brings about the expression ofnucleic acid molecules according to the invention, so that a finechemical is produced. In a preferred embodiment, the process furthermorecomprises the step of obtaining a cell comprising such nucleic acidsequences according to the invention, wherein a cell is transformed witha desaturase nucleic acid sequence, a gene construct or a vector whichbring about the expression of a desaturase nucleic acid according to theinvention, alone or in combination. In a further preferred embodiment,this method furthermore comprises the step of obtaining the finechemical from the culture. In an especially preferred embodiment, thecell belongs to the order of the ciliates, to microorganisms such asfungi, or to the plant kingdom, in particular to oil crop plants;especially preferred are microorganisms or oil crop plants, for examplepeanut, oilseed rape, canola, linseed, soybean, safflower (thistle),sunflowers or borage.

Transgenic/recombinant is to be understood as meaning, for the purposesof the invention, that the nucleic acids according to the invention arenot at their natural location in the genome of an organism; it ispossible here for the nucleic acids to be expressed homologously orheterologously. However, transgenic/recombinant also means that thenucleic acids according to the invention are at their natural locationin the genome of an organism, but that the sequence has been modifiedover the natural sequence and/or that the regulatory sequences of thenatural sequences have been modified. Transgenic/recombinant preferablydescribes the expression of the nucleic acids according to the inventionat an unnatural location in the genome, that is to say a homologous or,preferably, heterologous expression of the nucleic acids exists.Preferred transgenic organisms are the abovementioned transgenic plants,preferably oil crop plants.

The polyunsaturated fatty acids contained in the fatty acid estersproduced by the method according to the invention can be liberated, forexample, via treatment with alkali such as aqueous KOH or NaOH,advantageously in the presence of an alcohol such as methanol orethanol, and can be isolated via, for example, phase separation andsubsequent acidification, with, for example, H₂SO₄.

A further subject matter of the invention are oils, lipids and/or fattyacids containing at least two double bonds in the fatty acid molecules,preferably three, four, five or six double bonds, which have beenproduced by the above-described method according to the invention. Afurther subject matter of the invention are also compositions comprisingthe abovementioned oils, lipids and/or fatty acids, and the use of theoils, lipids and/or fatty acids or of the compositions in feed,foodstuffs, cosmetics or pharmaceuticals.

A further aspect of the invention relates to methods of modulating theproduction of a molecule by a microorganism. These methods encompass thecontacting of the cell with a substance which modulates the desaturaseactivity according to the invention alone or in combination or thedesaturase nucleic acid expression, so that a cell-associated activityis modified in relation to the same activity in the absence of thesubstance. In a preferred embodiment, one or two metabolic pathway(s) ofthe cell for lipids and fatty acids, cofactors and enzymes is/aremodulated, or the transport of compounds through these membranes ismodulated, so that the yield or the production rate of a desired finechemical by this microorganism is improved. The substance whichmodulates the desaturase activity can be a substance which stimulatesthe desaturase activity or desaturase nucleic acid expression or whichcan be used as intermediate in fatty acid biosynthesis. Examples ofsubstances which stimulate the desaturase activity or desaturase nucleicacid expression are, inter alia, small molecules, active desaturases anddesaturase-encoding nucleic acids which have been introduced into thecell. Examples of substances which inhibit the desaturase activity ordesaturase expression are, inter alia, small molecules and antisensedesaturase nucleic acid molecules.

A further aspect of the invention relates to methods of modulating theyields of a desired compound from a cell, comprising introducing, into acell, a wild-type or mutant desaturase gene which is either maintainedon a separate plasmid or integrated into the genome of the host cell.Upon integration into the genome, integration can be random or can beeffected by recombination in such a way that the native gene is replacedby the copy introduced, thereby modulating the production of the desiredcompound by the cell, or by using a gene in trans, so that the gene islinked operably to a functional expression unit comprising at least onesequence which ensures the expression of a gene and at least onesequence which ensures the polyadenylation of a functionally transcribedgene.

In a preferred embodiment, the yields are modified. In a furtherpreferred embodiment, the desired chemical is augmented, it beingpossible to reduce undesired interfering compounds. In an especiallypreferred embodiment, the desired fine chemical is a lipid or a fattyacid, a cofactor or an enzyme. In an especially preferred embodiment,this chemical is a polyunsaturated fatty acid. More preferably, it isselected from among arachidonic acid (ARA), eicosapentaenoic acid (EPA)or docosahexaenoic aicd (DHA).

The present invention provides novel nucleic acid molecules which aresuitable for identifying and isolating desaturases of PUFA biosynthesisand which can be used for the modification of oils, fatty acids, lipids,lipid-derived compounds and, most preferably, for the production ofpolyunsaturated fatty acids.

The invention furthermore provides multiexpression cassettes andconstructs for the multiparallel seed-specific expression of genecombinations in plants.

Microorganisms such as Crypthecodinium, Thraustochytrium, Phaeodactylumand Mortierella, Entomophthora and Mucor, and other algae and fungi andplants, in particular oil crop plants, are preferred organisms for themethod according to the invention.

Cloning vectors and techniques for the genetic manipulation of theabovementioned microorganisms and ciliates, algae or related organsismssuch as Phaeodactylum tricornutum, are described in WO 98/01572 or inFalciatore et al., 1999, Marine Biotechnology 1(3):239-251, and Dunahayet al., 1995, Genetic transformation of diatoms, J. Phycol. 31:1004-1012and the references cited therein. Thereby, the abovementioned nucleicacid molecules can be used in the method according to the invention bymodifying the organisms by means of genetic engineering so that theybecome better or more efficient producers of one or more fine chemicals.This improved production or production efficiency of a fine chemical canbe brought about by a direct effect of the manipulation of a geneaccording to the invention or by an indirect effect of thismanipulation. Fine chemicals for the purposes of the invention areunderstood as being fatty acid esters containing polyunsaturated fattyacids with at least two double bonds, such as sphingolipids,phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerides, diacyl glycerides, triacyl glycerides or other fatty acidesters containing the polyunsaturated fatty acids with at least twodouble bonds. They are furthermore understood as being compounds such asvitamins, for example vitamin E, vitamin C, vitamin B2, vitamin B6,pantolactone, carotenoids such as astaxanthin, β-carotene, zeaxanthinand others.

Mosses and algae are the only plant systems known which producesubstantial amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic aicd (EPA) and/or docosahexaenoicacid (DHA). Mosses contain PUFAs in membrane lipids, while algae,organisms related to algae and some fungi also accumulate substantialamounts of PUFAs in the triacylglycerol fraction. Nucleic acid moleculeswhich are isolated from such strains which also accumulate PUFAs in thetriacylglycerol fraction are therefore particularly advantageouslysuitable for modifying the lipid and PUFA production system in a host,in particular in microorganisms, such as in the microorganisms mentionedabove, and plants such as oil crop plants, for example oilseed rape,canola, linseed, soybean, sunflowers, borage. They can therefore be usedadvantageously in the method according to the invention.

The nucleic acids according to the invention are therefore particularlyadvantageously suitable for isolating nucleic acids fromtriacylglycerol-accumulating microorganisms and for identifying such DNAsequences and the enzymes encoded by them in other species which aresuitable for modifying the biosynthesis of PUFA-precursor molecules inthe organisms in question.

Microorganisms such as Crypthecodinium cohnii, Thraustochytrium andPhaeodactylum species are microorganisms which are capable ofaccumulating PUFAs such as ARA, EPA or DHA in triacylglycerols.Thraustochytria are phylogenetically also closely related to strains ofSchizochytria. The ability to identify desaturases with reference to thenucleic acids according to the invention, for example predicting thesubstrate specificity of enzymes, can therefore be of enormoussignificance. Furthermore, these nucleic acid molecules can act asreference sequences for mapping related genomes or for deriving PCRprimers.

The nucleic acid molecules according to the invention encode proteinstermed desaturases. These desaturases can exert, for example, a functioninvolved in the metabolism (for example in the biosynthesis or thedegradation) of compounds required for the synthesis of lipids or fattyacids, such as PUFAs, or can participate in the transmembrane transportof one or more lipid/fatty acid compounds, either into the cell or outof the cell.

The nucleic acid sequences according to the invention encode desaturaseswhich are suitable for the production of long-chain polyunsaturatedfatty acids, preferably having more than sixteen, eighteen or twentycarbon atoms in the carbon skeleton of the fatty acid and/or at leasttwo double bonds in the carbon chain, a nucleic acid according to theinvention encoding an enzyme capable of introducing double bonds at theΔ5 position, in another case at the Δ6 position and in a further case atthe Δ12 position. Large amounts of PUFAs may be obtained in thetriacylglycerol fraction with the aid of these nucleic acids.Furthermore, further desaturases have been isolated which, alone ortogether with a Δ4 desaturase, can be utilized for a method for theproduction of polyunsaturated fatty acids. In the application, thesingular, i.e. a desaturase gene or protein, is also understood asmeaning the plural, i.e. the desaturase genes or proteins.

The production of a trienoic acid with C₁₈-carbon chain with the aid ofdesaturases has already been demonstratead. However, in these methodsknown from the literature, the production of γ-linolenic acid wasclaimed. As yet, however, nobody has been able to demonstrate theproduction of very long-chain polyunsaturated fatty acids (with C₂₀carbon chain and longer and of trienoic acids and higher unsaturatedtypes) by modified organisms alone.

To produce the long-chain PUFAs according to the invention, thepolyunsaturated C₁₈-fatty acids must first be elongated by at least twocarbon atoms by the enzymatic activity of an elongase. Following anelongation cycle, this enzyme activity leads to C₂₀-fatty acids, andafter two, three and four elongation cycles, to C₂₂-, C₂₄- or C₂₆-fattyacids. The nucleic acid sequences disclosed in the present inventionwhich encode various desaturases can, in concert with elongases, lead tovery long-chain polyunsaturated fatty acids. The activity of thedesaturases according to the invention preferably leads to C₁₈-, C₂₀-and/or C₂₂-fatty acids with at least two double bonds in the fatty acidmolecule, preferably with three, four, five or six double bonds,especially preferably to C₁₈- and/or C₂₀-fatty acids with at least twodouble bonds in the fatty acid molecule, preferably with three, four orfive double bonds in the molecule. The elongation of the fatty acid canbe effected by combining the desaturases according to the invention withan elongase activity, it being possible to use the elongase encoded bySEQ ID NO: 9 in an advantageous fashion. After the elongation by theenzyme(s) according to the invention has taken place, furtherdesaturation steps such as, for example, a desaturation at the Δ5position, may take place. The combination with other elongases such asthose which lead to an elongation from C₁₈- to C₂₀-chains or from C₂₀-to C₂₂₋₂₄-chains as disclosed in WO 00/12720 may also be used and/or adesaturase with activity for the Δ4 position can advantageously beemployed in order to obtain the highly desaturated fatty acids. Theproducts of the desaturase activities and the possible furtherdesaturation therefore lead to preferred PUFAs with a higher degree ofdesaturation, such as dihomo-γ-linolenic acid, docosadienoic acid,arachidonic acid, ω6-eicosatrienedihomo-γ-linolenic acid,eicosapentaenoic acid, ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid,docosapentaenoic acid or docosahexaenoic acid. Substrates of the enzymeactivity according to the invention are, for example, taxoleic acid;6,9-octadecadienoic acid, linoleic acid, pinolenic acid, α- orγ-linolenic acid or stearidonic acid and arachidonic acid,eicosatetraenoic acid, docosopentaenoic acid, eicosapentaenoic acid.Preferred substrates are linoleic acid, γ-linolenic acid and/ora-linolenic acid and arachidonic acid, eicosatetraenoic acid,docosapentaenoic acid, eicosapentaenoic acid. Especially preferredproducts of the process according to the invention are arachidonic acid,docosapentaenoic acid, eicosapentaenoic acid. The C₁₈-fatty acids withat least two double bonds in the fatty acid can be elongated by theenzymatic activity according to the invention in the form of the freefatty acid or in the form of the esters, such as phospholipids,glycolipids, sphingolipids, phosphoglycerides, monoacyl glycerides,diacyl glycerides or triacyl glycerides.

Of particular importance for human nutrition is the conjugated linoleicacid “CLA”. CLA is understood as meaning in particular fatty acids suchas C18:2^(9 cis, 11trans) or the isomer C18:2^(10trans, 12 cis), which,once taken up, can be desaturated or elongated in the body owing tohuman enzyme systems and can contribute to health-promoting effects. Thedesaturases according to the invention (Δ12-desaturase) also allow thoseconjugated fatty acids with at least two double bonds in the molecule tobe desaturated and thus allow such health-promoting fatty acids to bemade available for human nutrition. Further examples of conjugated fattyacids are α-parinaric acid, punicic acid, eleostearic acid andcalendulic acid.

Using cloning vectors in plants and in the transformation of plants likethose which are published and cited in: Plant Molecular Biology andBiotechnology (CRC Press, Boca Raton, Fla.), Chapter 6/7, pp. 71-119(1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, Eds.: Kung andR. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,Eds.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu.Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)), the nucleicacids according to the invention can be used for the recombinantmodification of a broad spectrum of plants so that this plant becomes abetter or more efficient producer of one or more lipid-derived products,such as PUFAs. This improved production or production efficiency of alipid-derived product, such as PUFAs, can be brought about by a directaction of the manipulation or an indirection action of thismanipulation.

A series of mechanisms exist by means of which the modification of adesaturase protein according to the invention can have a direct effecton the yield, production and/or production efficiency of a fine chemicalfrom an oil crop plant or a microorganism, owing to a modified protein.The number or activity of the desaturase protein or desaturase gene andof gene combinations of desaturases and elongases can be increased, sothat larger amounts of these compounds are produced de novo since theorganisms lacked this activity and ability to biosynthesize them priorto introduction of the gene in question. This also applies analogouslyto the combination with further desaturases or elongases or furtherenzymes of the lipid metabolism. The use of various divergent sequences,i.e. sequences which differ at the DNA sequence level, may also beadvantageous, or else the use of promoters for gene expression whichmakes possible a different temporal gene expression, for example as afunction of the degree of maturity of the seed or oil-storing tissue.

The introduction of a desaturase gene according to the invention, orseveral saturase genes, into an organism, alone or in combination withother genes in a cell can not only increase the biosynthesis flux towardthe end product, but also increase, or generate de novo, thecorresponding triacylglycerol composition. Likewise, the number oractivity of other genes which participate in the import of nutrientsrequired for the biosynthesis of one or more fine chemicals (for examplefatty acids, polar and neutral lipids) can be increased, so that theconcentration of the precursors, cofactors or intermediates within thecells or within the storage compartment is increased, thus furtherincreasing the ability of the cells to produce PUFAs as describedhereinbelow. Fatty acids and lipids themselves are desirable as finechemicals; by optimizing the activity or increasing the number of one ormore desaturases which participate in the biosynthesis of thesecompounds, or by destroying the activity of one or more desaturaseswhich participate in the breakdown of these compounds, it can bepossible to increase the yield, production and/or production efficiencyof fatty acid and lipid molecules from plants or microorganisms.

The mutagenesis of the desaturase gene(s) according to the invention mayfurthermore lead to a desaturase protein with modified activities whichhave a direct or indirect effect on the production of one or moredesired fine chemicals. For example, the number or activity of thedesaturase gene(s) according to the invention may be increased so thatthe normal metabolic wastes or by-products of the cell (possiblyincreased in quantity due to the overproduction of the desired finechemical) are exported efficiently before they can damage othermolecules or processes within the cell (which would decrease theviability of the cell) or to interfere with the fine chemicalbiosynthetic pathways (which would decrease the yield, production orproduction efficiency of the desired fine chemical). Furthermore, therelatively large intracellular quantities of the desired fine chemicalmay themselves be toxic to the cell or interfere with enzyme feedbackmechanisms, such as allosteric regulation; for example, by increasingthe activity or number of other downstream enzymes or detoxificationenzymes of the PUFA pathway, it might increase the allocation of thePUFA to the triacylglycerol fraction; the viability of seed cells mightbe increased, in turn leading to better development of cultured cells orto seeds which produce the desired fine chemical. The desaturaseaccording to the invention may also be manipulated in such a way thatthe relative amounts of the various lipid and fatty acid molecules areproduced. This may have a profound effect on the lipid composition ofthe membrane of the cell and generates novel oils in addition to theoccurrence of newly-synthesized PUFAs. Since each type of lipid hasdifferent physical properties, a change in the lipid composition of amembrane may significantly alter membrane fluidity Changes in membranefluidity can have an effect on the transport of molecules across themembrane and on the integrity of the cell, both of which have a profoundeffect on the production of fine chemicals. In plants, these changes mayadditionally impact on other traits, such as tolerance to abiotic andbiotic stress situations.

Biotic and abiotic stress tolerance is a general trait which it isdesired to transmit to a broad spectrum of plants such as maize, wheat,rye, oats, triticale, rice, barley, soybeans, peanut, cotton, linseed,flax, oilseed rape and canola, cassava, pepper, sunflower and Tagetes,solanaceous plants such as potato, tobacco, egg-plant and tomato, Viciaspecies, pea, alfalfa, bush plants (coffee, cacao, tea), Salix species,trees (oil palm, coconut) and perennial grasses and fodder crops. Beinga further embodiment of the invention, these crops are also preferredtarget plants for genetic engineering. Especially preferred plantsaccording to the invention are oil crop plants such as soybean, peanut,oilseed rape, canola, sunflower, linseed, safflower, trees (oil palm,coconut) or crops such as maize, wheat, rye, oats, triticale, rice,barley, alfalfa, or bush plants (coffee, cacao, tea).

Accordingly, an aspect of the invention relates to isolated nucleic acidmolecules (for example cDNAs) encompassing nucleotide sequences whichencode one desaturase or several desaturases or biologically activeparts thereof, or nucleic acid fragments which are suitable as primersor hybridization probes for detecting or amplifying desaturase-encodingnucleic acids (for example DNA or mRNA). In especially preferredembodiments, the nucleic acid molecule encompasses one of the nucleotidesequences shown in sequence ID NO:1 or 3 and 5 or the coding region or acomplement of one of these nucleotide sequences. In other especiallypreferred embodiments, the isolated nucleic acid molecule according tothe invention encompasses a nucleotide sequence which hybridizes with anucleotide sequence as shown in the sequence SEQ ID NO: 1, 3, 5 or 11 ora portion thereof or which has at least 50% homology, preferably atleast approximately 60% homology, more preferably at least approximately70%, 80% or 90% homology and very especially preferably at leastapproximately 95%, 96%, 97%, 98%, 99% or more homology therewith. Inother preferred embodiments, the isolated nucleic acid molecule encodesone of the amino acid sequences shown in sequence SEQ ID NO: 2, 4, 6 or12. The preferred desaturase gene according to the invention preferablyalso has at least one of the desaturase activities described herein.

In a further embodiment, the isolated nucleic acid molecule encodes aprotein or a portion thereof, the protein or the portion thereofcomprising an amino acid sequence which has sufficient homology with anamino acid sequence of the sequence SEQ ID NO: 2, 4, 6 or 12 that theprotein or the portion thereof retains a desaturase activity.Preferably, the protein or the portion thereof encoded by the nucleicacid molecule retains the ability of participating in the metabolism ofcompounds required for the synthesis of cell membranes of plants or inthe transport of molecules across these membranes. In one embodiment,the protein encoded by the nucleic acid molecule has at leastapproximately 50% homology, preferably at least approximately 60%homology, more preferably at least approximately 70%, 80% or 90% andvery especially preferably at least approximately 95%, 96%, 97%, 98%,99% or more homology with an amino acid sequence of the sequence SEQ IDNO: 2, 4, 6 or 12. In a further preferred embodiment, the protein is afull-length protein which is essentially in parts homologous to acomplete amino acid sequence of SEQ ID NO: 2, 4, 6 or 12 (which isderived from the open reading frame shown in SEQ ID NO: 1, 3, 5 or 11).

In other embodiments, the isolated desaturase encompasses an amino acidsequence which has at least approximately 50% homology with one of theamino acid sequences of SEQ ID NO: 2, 4, 6 or 12 and which canparticipate in the metabolism of compounds required for the synthesis offatty acids in a microorganism or plant cell or in the transport ofmolecules across these membranes, desaturated C₁₈- or C₂₀₋₂₂-carbonchains being understood with double bonds in at least two positions.

In another preferred embodiment, the isolated nucleic acid moleculeoriginates from Phaeodactylum tricornutum UTEX646 and encodes a protein(for example a desaturase fusion protein) containing a biologicallyactive domain which has at least approximately 50% or more homology withan amino acid sequence of the sequence SEQ ID NO: 2, 4, 6 or 12 andretains the ability of participating in the metabolism of compoundsrequired in the synthesis of cell membranes of plants or in thetransport of molecules across these membranes or has at least one of thedesaturation activities resulting in PUFAs such as GLA, ALA,dihomo-γ-linolenic acid, ARA, EPA or DHA or their precursor molecules,and also encompasses heterologous nucleic acid sequences which encode aheterologous polypeptide or regulatory proteins.

As an alternative, the isolated desaturase can comprise an amino acidsequence which is encoded by a nucleotide sequence which hybridizes witha nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, for example understringent conditions, or which has at least approximately 50% homology,preferably at least approximately 60% homology, more preferably at leastapproximately 70%, 80% or 90% homology and even more preferably at leastapproximately 95%, 96%, 97%, 98%, 99% or more homology therewith. It isalso preferred for the preferred desaturase forms likewise to have oneof the desaturase activities described herein.

In another embodiment, the isolated nucleic acid molecule is at least15, 25, 50, 100, 250 or more nucleotides in length and hybridizes understringent conditions with a nucleic acid molecule comprising anucleotide sequence of SEQ ID NO: 1, 3, 5 or 17. Preferably, theisolated nucleic acid molecule corresponds to a naturally occurringnucleic acid molecule. More preferably, the isolated nucleic acidmolecule encodes naturally occurring Phaeodactylum desaturase or abiologically active portion thereof.

A further embodiment of the invention are expression cassettes whichmake possible the expression of the nucleic acids according to theinvention with the sequences SEQ ID NO: 1, 3, 5 or 11 in the variousorganisms such as microorganisms, for example bacteria, fungi, yeasts,ciliates, algae or animal or plant cells, or in animals or plants.

The expression cassette (=nucleic acid construct or fragment) accordingto the invention is to be understood as meaning the sequences mentionedin SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11 which arethe result of the genetic code and/or the functional or nonfunctionalderivatives thereof which had been linked functionally to one or moreregulatory signals for advantageously increasing gene expression andwhich govern the expression of the coding sequence in the host cell.These regulatory sequences are intended to make possible the targetedexpression of the genes and of protein expression. Depending on the hostorganism, this may mean, for example, that the gene is first induced andonly then expressed and/or overexpressed, or that it is expressed and/oroverexpressed immediately. For example, these regulatory sequences aresequences to which inductors or repressors bind, thus regulating theexpression of the nucleic acid. In addition to these novel regulatorysequences, or instead of these sequences, the natural regulation ofthese sequences before the actual structural genes may still be presentand, if appropriate, may have been genetically modified so that thenatural regulation has been eliminated and gene expression increased.However, the gene construct may also have a simpler construction, thatis to say no additional regulatory sequences were inserted before thenucleic acid sequence or its derivatives, and the natural promotertogether with its regulation has not been removed. Instead, the naturalregulatory sequence was mutated in such a way that regulation no longertakes place and/or gene expression is increased. These modifiedpromoters can be inserted before the natural gene in the form ofpart-sequences (=promoter with parts of the nucleic acid sequencesaccording to the invention) or else alone, in order to increase theactivity. Moreover, the gene construct can additionally advantageouslyalso comprise one or more of what are known as enhancer sequences linkedfunctionally to the promoter, and these make possible an increasedexpression of the nucleic acid sequence. Additional advantageoussequences, such as further regulatory elements or terminators, may alsobe inserted at the 3′ end of the DNA sequences. The Δ5 desaturase/Δ6desaturase and/or Δ12 desaturase genes may be present in the expressioncassette (=gene construct) in one or more copies.

As described above, the regulatory sequences or factors can preferablyhave a positive effect on the gene expression of the introduced gene,thus increasing it. Thus, an enhancement of the regulatory elements canadvantageously take place at transcription level, by using strongtranscription signals such as promoters and/or enhancers. In addition,however, enhanced translation is also possible, for example by improvingthe stability of the mRNA.

A further aspect of the invention relates to vectors, for examplerecombinant expression vectors, comprising at least one nucleic acidmolecule according to the invention, and to host cells into which thesevectors have been introduced, in particular microorganisms, plant cells,plant tissues, plant organs or intact plants. In one embodiment, such ahost cell can store fine chemical compounds, in particular PUFAs; toisolate the desired compound, the cells are harvested. The compound(oils, lipids, triacyl glycerides, fatty acids) or the desaturase canthen be isolated from the medium or from the host cell which, in thecase of plants, are cells comprising or storing the fine chemicals, mostpreferably cells of storage tissues such as seed coats, tubers,epidermis cells and seed cells, endosperm or embryo tissue.

Yet another aspect of the invention relates to a genetically modifiedtransgenic plant, preferably an oil crop plant as mentioned above,especially preferably an oilseed rape or linseed plant into which adesaturase gene has been introduced. In one embodiment, the genome ofoilseed rape or linseed has been modified by introducing, as transgene,a nucleic acid molecule according to the invention which encodes awild-type or mutated desaturase sequence. In another embodiment, anendogenous desaturase gene in the genome of the donor organismPhaeodactylum has been destroyed functionally by mutagenesis anddetection by means of DNA sequences or has been repressed by means ofantisense technology. In a preferred embodiment, oilseed rape or linseedis also used for the production of a desired compound such as lipids andfatty acids, with PUFas being especially preferred.

In yet another preferred embodiment, the moss Physcomitrella patens canbe used for demonstrating the function of a desaturase gene usinghomologous recombination on the basis of the nucleic acids described inthe present invention.

Yet another aspect of the invention relates to an isolated desaturasegene or a part, for example a biologically active part, thereof. In apreferred embodiment, the isolated desaturase or a part thereof canparticipate in the metabolism of compounds required for the synthesis ofcell membranes in a microorganism or a plant cell or in the transport ofmolecules via its membranes. In a further preferred embodiment, theisolated desaturase or the part thereof has sufficient homology with anamino acid sequence of SEQ ID NO: 2, 4, 6 or 12 to retain the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes in microorganisms or plant cells or in the transport ofmolecules via these membranes.

The invention also provides an isolated preparation of a desaturase inthe form of a crude extract or as a pure protein.

The desaturase polypeptide or a biological active part thereof canadvantageously be linked functionally to a further polypeptide which hasan enzymatic activity other than the desaturases, for example anelongase, acyltransferase or other activity, to form a fusion protein.This fusion protein advantageously has an activity which differs fromthat of the desaturase alone. In other preferred embodiments, thisfusion protein participates in the metabolism of compounds which arerequired for the synthesis of lipids and fatty acids, cofactors andenzymes in microorganisms or plants, or in the transport of moleculesvia these membranes. Especially preferably, the introduction of thisfusion protein into a host cell modulates the production of a desiredcompound within a cell and by the cell. In a preferred embodiment, thesefusion proteins also contain Δ4-, Δ5- or Δ6-, Δ8-, Δ15-, Δ17- orΔ19-desaturase activities, alone or in combination. Preferredembodiments are, in particular, those gene combinations which areselected from among SEQ ID NO: 7 or 9, or parts thereof, derivatives ortheir homologs. Particularly preferred are those combinations whichcontain the complete protein activity as in SEQ ID NO: 1, 3, 5 or 11and, inserted into multiexpression cassettes defined by SEQ ID NO: 13,14, 15, 16 and 17, are suitable for the transformation of plants andexpression in plants.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to (an) isolated nucleic acid sequence(s) encodinga polypeptide with desaturase activity selected from the groupconsisting of

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,-   b) nucleic acid sequences which, owing to the degeneracy of the    genetic code, are obtained by backtranslating the amino acid    sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ    ID NO: 12,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encode    polypeptides with the amino acid sequences shown in SEQ ID NO: 2,    SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least 50%    homology at the amino acid level, without essentially reducing the    enzymatic action of the polypeptides.

The invention furthermore relates to (an) amino acid sequence(s) whichis/are encoded by the abovementioned nucleic acid sequence(s) (for thepurposes of the invention, the singular is intended to comprise theplural and vice versa). Specifically, the invention relates to aminoacid sequences encoded by the sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 or SEQ ID NO: 11.

The present invention provides nucleic acids and protein molecules withdesaturase activity which participate in the metabolism of lipids andfatty acids, PUFA cofactors and enzymes in the moss Physcomitrellapatens or in the transport of lipophilic compounds via membranes. Thecompounds according to the invention can be used for modulating theproduction of fine chemicals from organisms, for example microorganisms,such as ciliates, fungi, yeasts, bacteria, algae and/or plants such asmaize, wheat, rye, oats, triticale, rice, barley, soybean, peanut,cotton, Linum species such as linseed or flax, Brassica species such asoilseed rape, canola and turnip rape, pepper, sunflower, borage, eveningprimrose and Tagetes, Solanaceae plants such as potato, tobacco,egg-plant and tomato, Vicia species, pea, cassava, alfalfa, bush plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut) andperennial grasses and fodder crops, either directly (for example whenthe overexpression or optimization of a fatty acid biosynthesis proteinhas a direct effect on the yield, production and/or productionefficiency of the fatty acid from modified organisms) or they can havean indirect effect which nevertheless leads to an increased yield,production and/or production efficiency of the desired compound or to adecrease in undesired compounds (for example when the modulation of themetabolism of lipids and fatty acids, cofactors and enzymes leads tochanges in yield, production and/or production efficiency or thecomposition of the desired compound within the cells, which, in turn,may have an effect on the production of one or more fine chemicals).Aspects of the invention are illustrated in greater detail hereinbelow.

I. Fine Chemicals and PUFAs

The term “fine chemical” is known in the art and encompasses moleculeswhich have been produced by an organism and which are used in a varietyof industries such as, by way of example but not by way of limitation,the pharmaceuticals industry, agro industry, food industry and cosmeticsindustry. These compounds encompass lipids, fatty acids, cofactors andenzymes and the like (as described, for example, in Kuninaka, A. (1996)Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6,Rehm et al., Ed., VCH Weinheim and references cited therein), lipids,saturated and unsaturated fatty acids (for example arachidonic acid),vitamins and cofactors (as described in Ullmann's Encyclopedia ofIndustrial Chemistry, Vol. A27, Vitamins, pp. 443-613 (1996) VCHWeinheim and references cited therein; and Ong, A. S., Niki, E., &Packer, L. (1995) Nutrition, Lipids, Health and Disease Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research—Asia, held September1-3, 1994, in Penang, Malaysia, AOCS Press (1995)), enzymes and allother chemicals described by Gutcho (1983) in Chemicals by Fermentation,Noyes Data Corporation, ISBN: 0818805086, and references cited therein.The metabolism and the uses of certain fine chemicals are illustrated ingreater detail hereinbelow.

The combination of various precursor molecules and biosynthetic enzymesleads to the production of various fatty acid molecules, which has adecisive effect on membrane composition. It can be assumed that PUFAsare not only just incorporated into triacylglycerol, but also intomembrane lipids.

Membrane synthesis is a well characterized process in which a number ofcomponents, including lipids as part of the bilayer membrane, areinvolved. The production of novel fatty acids such as PUFAs cantherefore generate novel properties of membrane functions within a cellor an organism.

Cell membranes serve a multiplicity of functions in a cell. First andforemost, a membrane delimits the contents of a cell from theenvironment, thus imparting integrity to the cell. Membranes can alsoact as barriers against the influx of dangerous or undesired compoundsor else against the efflux of desired compounds.

For more detailed descriptions and involvements of membranes and themechanisms involved, see Bamberg, E., et al. (1993) Charge transport ofion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26:1-25; Gennis,R. B. (1989) Pores, Channels and Transporters, in: Biomembranes,Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; andNikaido, H., and Saier, H. (1992) Transport proteins in bacteria: commonthemes in their design, Science 258:936-942, and the citations containedin each of these references.

Lipid synthesis can be divided into two parts: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Customary lipids used in membranesencompass phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA either into malonyl-CoA by acetyl-CoA carboxylase or intoacetyl-ACP by acetyl transacylase. After a condensation reaction, thesetwo product molecules together form acetoacetyl-ACP, which is convertedvia a series of condensation, reduction and dehydration reactions togive a saturated fatty acid molecule with the desired chain length. Theproduction of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (as regards fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., pp. 612-636 and referencescontained therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes.Thieme: Stuttgart, N.Y., and the references contained therein, andMagnuson, K., et al. (1993) Microbiological Reviews 57:522-542 and thereferences contained therein).

Examples of precursors for PUFA biosynthesis are oleic acid, linoleicacid and linolenic acid. These C₁₈ carbon fatty acids must be elongatedto C₂₀ and C₂₂ to give fatty acids of the eicosa and docosa chain type.Various desaturases such as enzymes which have Δ12-desaturase,Δ15-desaturase, Δ6-desaturase, Δ5- and Δ4-desaturase activity, can leadto arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid andvarious other long-chain PUFAs which can be extracted and used forvarious purposes in food and feed, cosmetic or pharmaceuticalapplications.

To produce long-chain PUFAs, the polyunsaturated C₁₈- or C₂₀-fatty acidsmust be polydesaturated as mentioned above. The nucleic acid sequencesaccording to the invention encode first functionally active desaturasesfrom Phaeodactylum tricornutum, a microorganism comprising PUFAs in thetriacylglycerol fraction. Double bonds can be introduced into the Δ5, Δ6or Δ12 position with the desaturases according to the invention. Theactivities of the desaturases according to the invention preferably leadto C₁₈-+C₂₀-fatty acids with at least two, three, four or five doublebonds in the fatty acid molecule, preferably to C₂₀-fatty acids with,advantageously, three, four or five double bonds in the fatty acidmolecule. Desaturation can be effected before or after elongation of thefatty acid in question. The products of the desaturase activities and ofthe possible further desaturation and elongation therefore lead topreferred PUFAs with a higher degree of desaturation, including afurther elongation of C₂₀- to C₂₂-fatty acids, to fatty acids such aslinoleic acid, docosadienoic acid, dihomo-γ-linolenic acid, arachidonicacid, ω6-eicosatrienedihomo-γ-linolenic acid, eicosapentaenoic acid,ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoic acidor docosahexaenoic acid. Preferred substrates of this enzyme activityaccording to the invention are taxoleic acid, 6,9-octadecadienoic acid,oleic acid, linoleic acid, y-linolenic acid, pinolenic acid, α-linolenicacid, arachidonic acid, eicosapentaenoic acid or stearidonic acid.Preferred substrates are linoleic acid, γ-linolenic acid and/orα-linolenic acid, dihomo-γ-linolenic acid or arachidonic acid,eicosatetraenoic acid or eicosapentaenoic acid. The C₁₈- or C₂₀-fattyacids with at least two double bonds in the fatty acid can be elongatedby the enzyme activity according to the invention in the form of thefree acid or in the form of the esters, such as phospholipids,glycolipids, sphingolipids, phosphoglycerides, monoacyl glycerides,diacyl glycerides, triacyl glycerides or other esters.

Furthermore, fatty acids must subsequently be transported to variouslocations of modification and incorporated into the triacylglycerolstorage lipid. Another important step in lipid synthesis is the transferof fatty acids to the polar head groups, for example by glycerol fattyacid acyl transferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).

For publications on plant fatty acid biosynthesis, desaturation, lipidmetabolism and the membrane transport of fatty compounds,beta-oxidation, fatty acid modification and cofactors, triacylglycerolstorage and assembly including the references cited therein, see thefollowing articles: Kinney, 1997, Genetic Engeneering, Ed.: J K Setlow,19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklinand Cahoon, 1998, Annu Rev. Plant Physiol. Plant Mol. Biol. 49:611-641;Voelker, 1996, Genetic Engineering, Ed.: J K Setlow, 18:111-13;Gerhardt, 1992, Prog. Lipid R. 31:397-417; Gühnemann-Schäfer & Kindl,1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog.Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry andMolecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murataand Somerville, Rockville, American Society of Plant Physiologists,150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.

Vitamins, cofactors and nutraceuticals, such as PUFAs, encompass a groupof molecules which higher animals can no longer synthesize and thereforehave to take up, or which higher animals can no longer synthesizethemselves to a sufficient degree and must therefore take upadditionally, even though they are readily synthesized by otherorganisms such as bacteria. The biosynthesis of these molecules inorganisms which are capable of producing them, such as in bacteria, hasbeen largely characterized (Ullmann's Encyclopedia of IndustrialChemistry, “Vitamins”, Vol. A27, pp. 443-613, VCH Weinheim, 1996;Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry andMolecular Biology, John Wiley & Sons; Ong, A. S., Niki, E., & Packer, L.(1995) “Nutrition, Lipids, Health and Disease” Proceedings of theUNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research Asia, held September1-3, 1994, in Penang, Malaysia, AOCS Press, Champaign, Ill. X, 374 pp.).

The abovementioned molecules are either biologically active moleculesthemselves or precursors of biologically active substances which acteither as electron carriers or as intermediates in a multiplicity ofmetabolic pathways. Besides their nutritional value, these compoundsalso have a significant industrial value as colorants, antioxidants andcatalysts or other processing auxiliaries. (For a review over structure,activity and industrial applications of these compounds, see, forexample, Ullmann's Encyclopedia of Industrial Chemistry, “Vitamins”,Vol. A27, pp. 443-613, VCH Weinheim, 1996). Polyunsaturated fatty acidshave a variety of functions and health-promoting effects, for example inthe case of coronary heart disease, inflammatory mechanisms, children'snutrition and the like. For publications and references including thereferences cited therein, see: Simopoulos, 1999, Am. J. Clin. Nutr. 70(3rd Suppl.):560-569, Takahata et al., Biosc. Biotechnol. Biochem. 1998,62(11):2079-2085, Willich and Winther, 1995, Deutsche MedizinischeWochenschrift 120(7):229 et seq.

II. Elements and Processes of the Invention

The present invention is based, inter alia, on the discovery of novelmolecules termed herein desaturase nucleic acid and desaturase proteinmolecules, which exert an effect on the production of cell membranes andlipids in Phaeodactylum tricornutum and, for example, have an effect onthe movement of molecules via these membranes. In one embodiment, thedesaturase molecules participate in the metabolism of compounds requiredfor the synthesis of cell membranes in organisms, such as microorganismsand plants, or indirectly affect the transport of molecules via thesemembranes. In a preferred embodiment, the activity of the desaturasemolecules according to the invention for regulating the production ofmembrane components and membrane transport has an effect on theproduction of the desired fine chemical by this organism. In anespecially preferred embodiment, the activity of the desaturasemolecules according to the invention is modulated so that the yield,production and/or production efficiency of the metabolic pathways ofmicroorganisms or plants which regulate the desaturases according to theinvention are modulated and the transport efficiency of compoundsthrough the membranes is modified, which either directly or indirectlymodulates the yield, production and/or production efficiency of adesired fine chemical by microorganisms and plants.

The term “desaturase” or “desaturase polypeptide” encompasses proteinswhich participate in the desaturation of fatty acids. Examples ofdesaturases are disclosed in SEQ ID NO: 1, 3, 5, 11 or their homologues,derivatives or analogs. The terms desaturase or desaturase nucleic acidsequence(s) encompass nucleic acid sequences which encode a desaturaseand part of which can be a coding region and also corresponding 5′- and3′-untranslated sequence regions. Examples of desaturase genes are thoseshown in SEQ ID NO: 1, 3, 5 or 11. The terms production and productivityare known in the art and encompass the concentration of the fermentationproduct (for example of the desired fine chemical) which is formedwithin a specific period and in a specific fermentation volume (forexample kg product per hour per liter). The term production efficiencyencompasses the time required for achieving a particular productionquantity (for example the time required by the cell to establish itsparticular throughput rate of a fine chemical). The term yield orproduct/carbon yield is known in the art and encompasses the efficiencywith which the carbon source is converted into the product (i.e. thefine chemical). This is usually expressed as, for example, kg productper kg carbon source. Increasing the yield of production of the compoundincreases the amount of the molecules obtained or of the suitablemolecules of this compound obtained in a specific quantity of cultureover a defined period. The terms biosynthesis or biosynthetic pathwayare known in the art and encompass the synthesis of a compound,preferably of an organic compound, by a cell from intermediates, forexample in a multi-step process which is subject to strong regulation.The terms catabolism or catabolic pathway are known in the art andencompass the cleavage of a compound, preferably of an organic compound,by a cell into catabolytes (in general terms, smaller or less complexmolecules), for example in a multi-step process which is subject tostrong regulation. The term metabolism is known in the art andencompasses the totality of the biochemical reactions which take placein an organism. The metabolism of a certain compound (for example themetabolism of a fatty acid) thus encompasses the totality of thebiosynthetic, modification and catabolic pathways of this compound inthe cell which are relevant to this compound.

In another embodiment, the nucleic acid sequences according to theinvention which encode desaturase molecules can modulate the productionof a desired molecule, such as a fine chemical, in a microorganism or inplants. There exist a series of mechanisms by which the modification ofa sequence according to the invention can directly affect the yield,production and/or production efficiency of a fine chemical from amicroorganism strain or plant strain comprising this modified protein.The number or activity of desaturases participating in the transport ofmolecules of fine chemicals within, or out of, the cell can beincreased, so that greater amounts of these compounds are transportedvia membranes, from which they can be obtained and converted into eachother with greater ease. Furthermore, fatty acids, triacylglycerolsand/or lipids are desirable fine chemicals themselves; optimizing theactivity or increasing the number of one or more desaturases accordingto the invention which participate in the biosynthesis of thesecompounds, or by interfering with the activity of one or moredesaturases which participate in the catabolism of these compounds,makes increasing the yield, production and/or production efficiency offatty acid molecules and lipid molecules from organisms such asmicroorganisms or plants, possible.

The mutagenesis of the nucleic acid sequences according to the inventioncan give rise to desaturases with modified activities which indirectlyaffect the production of one or more desired fine chemicals frommicroorganisms or plants. For example, desaturases according to theinvention which participate in the export of waste products can exhibita greater number or higher activity, so that the normal metabolic wasteproducts of the cell (whose quantity might be increased owing to theoverproduction of the desired fine chemical) are exported efficientlybefore they can damage the molecules in the cell (which would reducecell viability) or interfere with the biosynthetic pathways of the finechemicals (which would reduce the yield, production or productionefficiency of a desired fine chemical). The relatively largeintracellular amounts of the desired fine chemical themselves canfurthermore be toxic to the cell, so that increasing the activity ornumber of transporters capable of exporting these compounds from thecell results in an increased viability of the cell in culture, which, inturn, leads to a higher number of cells in the culture which produce thedesired fine chemical. The desaturases according to the invention canalso be manipulated in such a way that the corresponding amounts ofdifferent lipid molecules and fatty acid molecules are produced. Thiscan have a substantial effect on the lipid concentration of the cellmembrane. Since each lipid type has different physical properties, amodification of the lipid composition of a membrane can significantlymodify membrane fluidity. Modifications of the membrane fluidity canaffect the transport of molecules via the membrane and cell integrity,each of which has a substantial effect on the production of finechemicals from microorganisms and plants in large-scale fermentationculture. Plant membranes impart specific properties such as tolerance tohigh and low temperatures, salt, drought and tolerance with respect topathogens such as bacteria and fungi. The modulation of the membranecomponents may therefore have a critical effect on the ability of theplants to survive under the abovementioned stress parameters. This cantake place via changes in signal cascades or directly via the modifiedmembrane composition (see, for example, Chapman, 1998, Trends in PlantScience, 3(11):419-426) and signal cascades (see Wang 1999, PlantPhysiology, 120:645-651) or affect the tolerance of low temperatures, asdisclosed in WO 95/18222.

The isolated nucleic acid sequences according to the invention arepresent, for example, in the genome of a Phaeodactylum tricomutumUTEX646 strain which is available via the algae collection of theUniversity of Texas, Austin.

The nucleotide sequence of the Phaeodactylum tricornutum cDNA and thederived amino acid sequences of the desaturases are shown in SEQ ID NO:1 to 6 and 11 and 12. Computer analyses were carried out which classifyand/or identify these nucleotide sequences as sequences which encodeproteins participating in the metabolism of cell membrane components orwhich participate in the transport of compounds via cell membranes, orof PUFA biosynthesis. ESTs with the database input NO: PT00107001OR andPT001078032R by the inventors constitute the sequences according to theinvention in SEQ ID NO: 1 and 3. The sequence of the fragment of ESTPT00107001OR was determined and is as shown in SEQ ID NO: 5. In asimilar manner, the sequence of clone PT001078032R is shown in SEQ IDNO: 1. Gene names were assigned to the clones. The abbreviations denote:Pp=Physcomitrella patens, Pt=Phaeodactylum tricomutum. PT001070010R ofSEQ ID NO: 5 encodes a novel gene which is homologous to Al2-desaturaseand PT001078032R encodes a novel Δ5-desaturase. Pt_des6 can be isolatedin accordance with Example 5a by means of polymerase chain reaction withthe aid of degenerate oligonucleotides. A fragment obtained in this waycan be isolated for screening a Phaeodactylum tricornutum cDNA library,and the coding region of a Phaeodactylum tricornutum Δ6-desaturase canbe obtained. A gene isolated in this way is termed Pt_des6 in Table 1and is shown in SEQ ID NO: 3. The corresponding amino acid sequences areobtained by translating the genetic code of sequence ID NO: 1, 3 and 5and are defined as SEQ ID NO: 2, 4 and 6 (see also Table 1). A furthernucleic acid sequence which encodes a Δ12-desaturase can also be foundin Table 1. It has the clone number PT001072031R.

TABLE 1 Nucleic acid SEQ Polypeptide Gene name Clone name ID NO: SEQ IDNO: Δ5-desaturase Pt_des5 PT001078032R 1 2 Δ6-desaturase Pt_des6 Pt_des63 4 Δ12-desaturase Pt_des12 PT001070010R 5 6 Δ6-desaturase Pp_des6Pp_des6 7 8 Δ6-elongase Pp_PSE1 PP001019019F 9 10 Δ12-desaturase Ptdes12.2 PT001072013R 11 12

The present invention also relates to proteins with an amino acidsequence which is essentially homologous with an amino acid sequence ofSEQ ID NO:2, 4, 6 or 12. As used in the present context, a protein withan amino acid sequence which is essentially homologous with a selectedamino acid sequence has at least approximately 50% homology with theselected amino acid sequence, for example the complete amino acidsequence selected. A protein with an amino acid sequence which isessentially homologous with a selected amino acid sequence can also haveat least approximately 50 to 60% homology, preferably at leastapproximately 60 to 70% homology and more preferably at leastapproximately 70 to 80%, 80 to 90% or 90 to 95% homology and mostpreferably at least approximately 96%, 97%, 98%, 99% or more homologywith a selected amino acid sequence.

The desaturase according to the invention or the biologically activepart or the fragment thereof can participate in the metabolism of lipidsrequired for the synthesis of membranes or storage lipids inmicroorganisms and can, in combination with further genes, in particularthose with elongase activity, contribute to activities required for theelongation of C₁₈- or C₂₀₋₂₂-PUFAs so that C₁₈-, C₂₀-, C₂₂- or C₂₄-PUFAsand related PUFAs are obtained. In this context, desaturases accordingto the invention can be cloned in combination with elongases and otherdesaturases in expression cassettes according to the invention andemployed for the transformation of plants with the aid of Agrobacterium.

Various aspects of the invention are described in greater detail in thesubsections which follow.

A. Isolated Nucleic Acid Molecules

One embodiment of the invention are isolated nucleic acids derived fromPUFA-producing microorganisms and encoding polypeptides which desaturateC₁₈-or C₂₀₋₂₂-fatty acids with at least one, two, three or four doublebonds in the fatty acid.

A further embodiment according to the invention are isolated nucleicacids encompassing nucleotide sequences encoding polypeptides whichdesaturate C₁₈- or C₂₀-fatty acids with at least one, two, three or fourdouble bonds in the fatty acid and which are selected from the groupconsisting of

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11,-   b) nucleic acid sequences which, owing to the degeneracy of the    genetic code, are obtained by backtranslating the amino acid    sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ    ID NO: 12,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1,    SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which encode    polypeptides with the amino acid sequences shown in SEQ ID NO: 2,    SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 and have at least 50%    homology at the amino acid level, without essentially reducing the    enzymatic action of the polypeptides.

The abovementioned nucleic acid according to the invention is derivedfrom organisms such as ciliates, fungi, algae or dinoflagellates whichare capable of synthesizing PUFAs, preferably from Phaeodactylumtricornutum or closely related organisms.

One aspect of the invention relates to isolated nucleic acid moleculeswhich encode desaturase polypeptide or biologically active partsthereof, and to nucleic acid fragments which suffice for use ashybridization probes or primers for identifying or amplifying adesaturase-encoding nucleic acid (for example desaturase DNA). The term“nucleic acid molecule” as used in the present context is intended toencompass DNA molecules (for example cDNA or genomic DNA) and RNAmolecules (for example mRNA) and DNA or RNA analogs which are generatedby means of nucleotide analogs. This term additionally encompasses theuntranslated sequence on the 3′ and the 5′ ends of the coding generegion: at least 500, preferably 200, especially preferably 100,nucleotides of the sequence upstream of the 5′ end of the coding regionand at least 100, preferably 50, especially preferably 20, nucleotidesof the sequence downstream of the 3′ end of the coding gene region. Thenucleic acid molecule can be single- or double-stranded, but ispreferably double-stranded DNA. An “isolated” nucleic acid molecule isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. An “isolated” nucleic acidpreferably has no sequences which naturally flank the nucleic acid inthe genomic DNA of the organism from which the nucleic acid is derived(for example sequences located at the 5′ and 3′ ends of the nucleicacid). In various embodiments, the isolated desaturase nucleic acidmolecule can comprise, for example, less than approximately 5 kb, 4 kb,3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in the genomic DNA of the cellfrom which the nucleic acid is derived (for example a Physcomitrellapatens cell). An “isolated” nucleic acid molecule, such as a cDNAmolecule, can moreover be essentially free from other cellular materialor culture medium if it is generated by recombinant techniques, or freefrom chemical precursors or other chemicals if it is synthesizedchemically.

A nucleic acid molecule according to the invention, for example anucleic acid molecule with a nucleotide sequence of SEQ ID NO:1 or apart thereof, can be isolated using standard techniques of molecularbiology and the sequence information provided herein. Also, for examplea homologous sequence or homologous, conserved sequence regions can beidentified at DNA or amino acid level with the aid of alignmentalgorithms. For example, a Phaeodactylum tricornutum cDNA can beisolated from a Phaeodactylum tricornutum library by using the completeSEQ ID NO:1, 3, 5 or 11 or a part thereof as hybridization probe andstandard hybridization techniques (as described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor,-N.Y., 1989). Moreover, a nucleic acid moleculeencompassing a complete sequence of SEQ ID NO: 1, 3, 5 or 11 or a partthereof can be isolated by polymerase chain reaction, whereoligonucleotide primers which are generated on the basis of thissequence or parts thereof, in particular regions around motifs ofExample 5a or modifications of the same in individual defined aminoacids are used (for example, a nucleic acid molecule encompassing thecomplete sequence of SEQ ID NO:1, 3, 5 or 11 or a part thereof can beisolated by polymerase chain reaction using oligonucleotide primerswhich have been generated on the basis of this same sequence of SEQ IDNO: 1, 3, 5 or 11). For example, mRNA can be isolated from cells (forexample by the guanidinium thiocyanate extraction method of Chirgwin etal. (1979) Biochemistry 18:5294-5299) and cDNA by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, availablefrom Seikagaku America, Inc., St.Petersburg, Fla.). Syntheticoligonucleotide primers for amplification by means of polymerase chainreaction can be generated on the basis of one of the sequences shown inSEQ ID NO: 1, 3, 5 or 11 and in FIG. 5 a or with the aid of the aminoacid sequences shown in SEQ ID NO: 2, 4, 6 or 12. A nucleic acidaccording to the invention can be amplified using cDNA or,alternatively, genomic DNA as template and suitable oligonucleotideprimers, in accordance with standard PCR amplification techniques. Thenucleic acid amplified in this way can be cloned into a suitable vectorand characterized by means of DNA sequence analysis. Oligonucleotideswhich correspond to a desaturase nucleotide sequence can be generated bystandard synthesis methods, for example using an automatic DNAsynthesizer.

The cDNA shown in SEQ ID NO: 1,3, 5 or 11 encompasses sequences whichencode desaturases (i.e. the “coding region”), and 5′-untranslatedsequences and 3′-untranslated sequences. Alternatively, the nucleic acidmolecule can only encompass the coding region of one of the sequences inSEQ ID NO: 1, 3, 5 or 11 or can comprise complete genomic fragmentswhich have been isolated from genomic DNA.

In a further preferred embodiment, an isolated nucleic acid moleculeaccording to the invention encompasses a nucleic acid molecule which isa complement of one of the nucleotide sequences shown in SEQ ID NO: 1,3, 5 or 11 or a part thereof. A nucleic acid molecule which iscomplementary to one of the nucleotide sequences shown in SEQ ID NO: 1,3, 5 or 11 is sufficiently complementary if it is capable of hybridizingwith one of the sequences stated in SEQ ID NO: 1, 3, 5 or 11, givingrise to a stable duplex.

Homologs of the novel desaturase nucleic acid sequences with thesequence SEQ ID NO: 1, 3, 5 or 11 means, for example, allelic variantswith at least approximately 50 to 60% homology, preferably at leastapproximately 60 to 70% homology, more preferably at least approximately70 to 80%, 80 to 90% or 90 to 95% homology and even more preferably atleast approximately 95%, 96%, 97%, 98%, 99% or more homology with one ofthe nucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 or theirhomologs, derivatives, analogs or parts thereof. In a further preferredembodiment, an isolated nucleic acid molecule according to the inventionencompasses a nucleotide sequences which hybridizes with one of thenucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 or a partthereof, for example under stringent conditions. Allelic variantsencompass, in particular, functional variants which can be obtained bythe deletion, insertion or substitution of nucleotides from/into thesequence shown in SEQ ID NO: 1, 3, 5 or 11, it being intended, however,for the enzyme activity of the resulting proteins which are synthesizedto be advantageously retained for the insertion of one or more genes.Proteins which retain the enzymatic activity of desaturase, that is tosay whose activity is essentially not reduced, means proteins with atleast 10%, preferably 20%, especially preferably 30%, very particularlypreferably 40%, of the original enzyme activity compared with theprotein encoded by SEQ ID NO: 2, 4, 6 or 12.

Homologs of SEQ ID NO: 1, 3, 5 or 11 also means, for example, bacterial,fungal and plant homologs, truncated sequences, single-stranded DNA orRNA of the coding and noncoding DNA sequence.

Homologs of SEQ ID NO: 1, 3, 5 or 11 also means derivatives such as, forexample, promoter variants. The promoters upstream of the nucleotidesequences stated can be modified by one or more nucleotidesubstitutions, by insertion(s) and/or deletion(s), without, however,interfering with the functionality or activity of the promoters. It isfurthermore possible for the activity of the promoters to be increasedby modifying their sequence or for them to be replaced completely bymore active promoters, even from heterologous organisms.

Moreover, the nucleic acid molecule according to the invention may onlyencompass part of the coding region of one of the sequences in SEQ IDNO: 1, 3, 5 or 11, for example a fragment which can be used as probe orprimer, or a fragment which encodes a biologically active segment of adesaturase. The nucleotide sequences determined from cloning thePhaeodactylum tricornutum desaturase gene allow the generation of probesand primers which are designed for identifying and/or cloning desaturasehomologs in other cell types and organisms and desaturase homologs fromother microalgae or related species. The probe/primer usuallyencompasses an essentially purified oligonucleotide. The oligonucleotideusually encompasses a nucleotide sequence region which hybridizes understringent conditions to at least approximately 12, preferablyapproximately 16, more preferably approximately 25, 40, 50 or 75successive nucleotides of a sense strand of one of the sequences statedin SEQ ID NO: 1, 3, 5 or 11, of an antisense strand of one of thesequences stated in SEQ ID NO: 1, 3, 5 or 11 or its homologs,derivatives or analogs or naturally occurring mutants thereof. Primersbased on a nucleoide sequence of SEQ ID NO: 1, 3, 5 or 11 can be used inPCR reactions for cloning desaturase homologs. Probes based on thedesaturase nucleotide sequences can be used for detecting transcripts orgenomic sequences which encode the same or homologous proteins. Inpreferred embodiments, the probe additionally encompasses a labelinggroup bound thereto, for example a radioisotope, a fluorescent compound,an enzyme or an enzyme cofactor. These probes can be used as part of atest kit for genomic markers for identifying cells which misexpress adesaturase, for example by measuring an amount of a desaturase-encodingnucleic acid in a cell sample, for example measuring the desaturase mRNAlevel, or for determining whether a genomic desaturase gene is mutatedor deleted.

In one embodiment, the nucleic acid molecule according to the inventionencodes a protein or part thereof which encompasses an amino acidsequence with sufficient homology with an amino acid sequence of SEQ IDNO: 2, 4, 6 or 12 for the protein or part thereof to retain the abilityto participate in the metabolism of compounds required for the synthesisof the cell membranes in microorganisms or plants or in the transport ofmolecules via these membranes. As used in the present context, the term“sufficient homology” refers to proteins or parts thereof whose aminoacid sequences have a minimum number of amino acid residues which areidentical with or equivalent to an amino acid sequence of SEQ ID NO:2(for example an amino acid residue with a similar side chain, such as anamino acid residue in one of the sequences of SEQ ID NO:2) so that theprotein or the part thereof can participate in the metabolism ofcompounds required for the synthesis of cell membranes in microorganismsor plants or in the transport of molecules via these membranes. Asdescribed herein, protein components of these metabolic pathways formembrane components or membrane transport systems can play a role in theproduction and secretion of one or more fine chemicals. Examples ofthese activities are also described herein. Thus, the “function of adesaturase” contributes either directly or indirectly to the yield,production and/or production efficiency of one or more fine chemicals.Examples of desaturase substrate specificity of the catalytic activityare stated in Tables 5 and 6.

In a further embodiment, derivatives of the nucleic acid moleculeaccording to the invention encode proteins with at least approximately50 to 60% homology, preferably at least approximately 60 to 70% homologyand more preferably at least approximately 70 to 80%, 80 to 90%, 90 to95% homology, and most preferably at least approximately 96%, 97%, 98%,99% or more homology with a complete amino acid sequence of SEQ ID NO:2.The homology of the amino acid sequence can be determined over theentire sequence region using the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153) or BESTFIT orGAP (Henikoff, S. and Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proc. Natl. Acad. Sci. USA 89:10915-10919.)

Parts of proteins encoded by the desaturase nucleic acid moleculesaccording to the invention are preferably biologically active parts ofone of the desaturases. As used herein, the term “biologically activepart of a desaturase” is intended to encompass a segment, for example adomain/motif, of a desaturase which can participate in the metabolism ofcompounds required for the synthesis of cell membranes in microorganismsor plants or in the transport of molecules via these membranes or whichhas an activity stated in Tables 5 and 6. An assay of the enzymaticactivity can be carried out in order to determine whether a desaturaseor a biologically active part thereof can participate in the metabolismof compounds required for the synthesis of cell membranes inmicroorganisms or plants or in the transport of molecules via thesemembranes. These assay methods as described in detail in Example 8 ofthe examples section are known to the skilled worker.

Additional nucleic acid fragments which encode biologically activesegments of a desaturase can be generated by isolating part of one ofthe sequences in SEQ ID NO: 1, 3, 5 or 11, expressing the encodedsegment of the desaturase or of the peptide (for example by recombinantexpression in vitro) and determining the activity of the encoded part ofthe desaturase or of the peptide.

Moreover, the invention encompasses nucleic acid molecules which differfrom one of the nucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11(and parts thereof) owing to the degeneracy of the genetic code andwhich thus encode the same desaturase as the one encoded by thenucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11. In anotherembodiment, an isolated nucleic acid molecule according to the inventionhas a nucleotide sequence which encodes a protein with an amino acidsequence shown in SEQ ID NO: 2, 4, 6 or 12. In a further embodiment, thenucleic acid molecule according to the invention encodes a full-lengthdesaturase protein which is essentially homologous to an amino acidsequence of SEQ ID NO: 2, 4, 6 or 12 (which is encoded by an openreading frame shown in SEQ ID NO: 1, 3, 5 or 11) and which can beidentified and isolated by customary methods.

In addition to the desaturase nucleotide sequence shown in SEQ ID NO: 1,3, 5 or 11, the skilled worker recognizes that DNA sequencepolymorphisms may exist which lead to changes in the amino acidsequences of the desaturases within a population (for example thePhaeodactylum tricornutum population). These genetic polymorphisms inthe desaturase gene can exist between individuals within a populationowing to natural variation. As used in the present context, the terms“gene” and “recombinant gene” refer to nucleic acid molecules with anopen reading frame which encodes a desaturase, preferably aPhaeodactylum tricornutum desaturase. These natural variants usuallycause a variance of 1 to 5% in the nucleotide sequence of the desaturasegene. All of these nucleotide variations and resulting amino acidpolymorphisms in desaturase which are the result of natural variationand do not alter the functional activity of desaturases are intended tocome within the scope of the invention.

Nucleic acid molecules which correspond to the natural variants andnon-Phaeodactylum-tricornutum-homologs, -derivatives and -analogs of thePhaeodactylum tricornutum cDNA can be isolated in accordance withstandard hybridization techniques under stringent hybridizationconditions owing to their homology with the Phaeodactylum tricornutumdesaturase nucleic acid disclosed herein using the Phaeodactylumtricornutum cDNA or part thereof as hybridization probe. In anotherembodiment, an isolated nucleic acid molecule according to the inventionhas a minimum length of 15 nucleotides and hybridizes under stringentconditions to the nucleic acid molecule which encompasses a nucleotidesequence of SEQ ID NO:1, 3, 5 or 11.

In other embodiments, the nucleic acid has a minimum length of 25, 50,100, 250 or more nucleotides. The term “hybridizes under stringentconditions” as used in the present context is intended to describehybridization and wash conditions under which nucleotide sequences whichhave at least 60% homology to each other usually remain hybridized toeach other. The conditions are preferably such that sequences which haveat least approximately 65% homology, more preferably approximately 70%homology and even more preferably at least approximately 75% or morehomology to each other usually remain hybridized to each other. Thesestringent conditions are known to the skilled worker and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989),6.3.1-6.3.6. A preferred, nonlimiting example of stringent hybridizationconditions are hybridizations in 6× sodium chloride/sodium citrate(=SSC) at approximately 45° C., followed by one or more wash steps in0.2×SSC, 0.1% SDS at 50 to 65° C. It is known to the skilled worker thatthese hybridization conditions differ depending on the type of nucleicacid and, for example, when organic solvents are present, with regard tothe temperature and the concentration of the buffer. The temperaturediffers, for example, under “standard hybridization conditions”depending on the type of the nucleic acid between 42° C. and 58° C. inaqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). If organicsolvent is present in the abovementioned buffer, for example 50%formamide, the temperature under standard conditions is approximately42° C. The hybridization conditions for DNA:DNA hybrids are preferablyfor example 0.1×SSC and 20° C. to 45° C., preferably between 30° C. and45° C. The hybridization conditions for DNA:RNA hybrids are preferablyfor example 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and55° C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid approximately 100 by (=base pairs) in lengthand a G+C content of 50% in the absence of formamide. The skilled workerknows how the hybridization conditions required can be determined withreference to textbooks, such as the one mentioned above, or from thefollowing textbooks: Sambrook et al., “Molecular Cloning”, Cold SpringHarbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press.

Preferably, an isolated nucleic acid molecule according to the inventionwhich hybridizes under stringent conditions to a sequence of SEQ IDNO:1, 3, 5 or 11 corresponds to a naturally occurring nucleic acidmolecule. As used in the present context, a “naturally occurring”nucleic acid molecule refers to an RNA or DNA molecule with a nucleotidesequence which occurs in nature (for example which encodes a naturalprotein). In one embodiment, the nucleic acid encodes a naturallyoccurring Phaeodactylum tricornutum desaturase.

In addition to naturally occurring variants of the desaturase sequencewhich may exist in the population, the skilled worker furthermorerecognizes that changes by means of mutation may also be introduced intoa nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, which leads tochanges in the amino acid sequence of the encoded desaturase withoutadversely affecting the functionality of the desaturase-protein. Forexample, nucleotide substitutions which lead to amino acid substitutionson “nonessential” amino acid residues can be generated in a sequence ofSEQ ID NO: 2, 4, 6 or 12. A “nonessential” amino acid residue is aresidue which can be altered in a wild-type sequence of one of thedesaturases (SEQ ID NO: 2, 4, 6 or 12) without altering, that is to sayessentially reducing, the activity of the desaturase, while an“essential” amino acid residue is required for the desaturase activity.Other amino acid residues (for example those which are not conserved, oronly semi-conserved, in the domain with desaturase activity), however,may not be essential for the activity and can therefore be modifiedwithout modifying the desaturase activity.

Accordingly, a further aspect of the invention relates to nucleic acidmolecules which encode desaturases comprising modified amino acidresidues which are not essential for the desaturase activity. Thesedesaturases differ from a sequence in SEQ ID NO: 2, 4, 6 or 12 withregard to the amino acid sequence while still retaining at least one ofthe desaturase activities described herein. In one embodiment, theisolated nucleic acid molecule encompasses a nucleotide sequenceencoding a protein, the protein encompassing an amino acid sequence withat least approximately 50% homology with an amino acid sequence of SEQID NO: 2, 4, 6 or 12 and being able to participate in the metabolism ofcompounds required for the synthesis of the cell membranes inPhaeodactylum tricornutum or in the transport of molecules via thesemembranes. The protein encoded by the nucleic acid molecule preferablyhas at least approximately 50 to 60% homology with one of the sequencesin SEQ ID NO:2, 4, 6 or 12, more preferably at least approximately 60 to70% homology with one of the sequences in SEQ ID NO:2, 4, 6 or 12, evenmore preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%homology with one of the sequences in SEQ ID NO: 2, 4, 6 or 12 and mostpreferably at least 96%, 97%, 98% or 99% homology with one of thesequences in SEQ ID NO: 2, 4, 6 or 12.

To determine the percentage homology of two amino acid sequences (forexample one of the sequences of SEQ ID NO: 2, 4, 6 or 12 and a mutatedform thereof) or of two nucleic acids, the sequences are written oneunderneath the other to allow optimum comparison (for example, gaps maybe introduced into the sequence of a protein or of a nucleic acid inorder to generate an optimal alignment with the other protein or theother nucleic acid). Then, the amino acid residues or nucleotides at thecorresponding amino acid positions or nucleotide positions are compared.If a position in a sequence (for example one of the sequences of SEQ IDNO: 2, 4, 6 or 12) is occupied by the same amino acid residue or thesame nucleotide as the corresponding position in the other sequence (forexample a mutated form of the sequence selected from SEQ ID NO: 2, 4, 6or 12), then the molecules are homologous at this position (i.e. aminoacid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”). The percentagehomology between the two sequences is a function of the number ofidentical positions which the sequences share (i.e. % homology=number ofidentical positions/total number of positions×100). The terms homologyand identity are thus to be considered as being synonymous.

An isolated nucleic acid molecule which encodes a desaturase which ishomologous with a protein sequence of SEQ ID NO: 2, 4, 6 or 12 can begenerated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11 sothat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoone of the sequences of SEQ ID NO: 1, 3, 5 or 11 by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are generated at oneor more of the predicted nonessential amino acid residues. In a“conservative amino acid substitution”, the amino acid residue isexchanged for an amino acid residue with a similar side chain. Familiesof amino acid residues with similar side chains have been defined in thespecialist field. These families encompass amino acids with basic sidechains (for example lysine, arginine, hystidine), acidic side chains(for example aspartic acid, glutamic acid), uncharged polar side chains(for example glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), unpolar side chains (for example alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (for example threonine, valine, isoleucine)and aromatic side chains (for example tyrosine, phenylalanine,tryptophan, histidine). A predicted nonessential amino acid residue in adesaturase is thus preferably exchanged for another amino acid residuefrom the same side-chain family. As an alternative, in anotherembodiment, the mutations can be introduced randomly over all or part ofthe desaturase-encoding sequence, for example by saturation mutagenesis,and the resulting mutants can be screened for the desaturase activity inorder to identify mutants which retain desaturase activity. Followingthe mutagenesis of one of the sequences of SEQ ID NO: 1, 3, 5 or 11, theencoded protein can be expressed recombinantly, and the activity of theprotein can be determined, for example using the assays described herein(see examples section).

In addition to the nucleic acid molecules which encode theabove-described desaturases, a further aspect of the invention relatesto isolated nucleic acid molecules which are “antisense” to the nucleicacid sequences according to the invention. An “antisense” nucleic acidencompasses a nucleotide sequence which is complementary to a “sense”nucleic acid which encodes a protein, for example complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can bind to asense nucleic acid by hydrogen bonds. The antisense nucleic acid can becomplementary to a complete desaturase-encoding strand or only to partthereof. In one embodiment, an antisense nucleotide acid molecule is“antisense” to a “coding region” of the coding strand of a nucleotidesequence encoding a desaturase. The term “coding region” refers to theregion of the nucleotide sequence which encompasses codons which aretranslated into amino acid residues (for example the entire codingregion which starts and ends with the stop codon, i.e. the last codonbefore the stop codon). In a further embodiment, the antisense nucleicacid molecule is “antisense” to a “noncoding region” of the codingstrand of a nucleotide sequence encoding desaturase. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region andare not translated into amino acids (i.e. which are also termed 5′- and3′-untranslated regions).

Given the desaturase-encoding sequences disclosed herein of the codingstrand (for example the sequences shown in SEQ ID NO: 1, 3, 5 or 11),antisense nucleic acids according to the invention can be designed inaccordance with the rules of Watson-Crick base pairing. The antisensenucleic acid molecule can be complementary to all of the coding regionof desaturase mRNA, but is more preferably an oligonucleotide which is“antisense” to only part of the coding or noncoding region of thedesaturase mRNA. The antisense oligonucleotide can be complementary, forexample, to the region around the translation start of desaturase mRNA.An antisense oligonucleotide can have a length of, for example,approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and morenucleotides. An antisense nucleic acid according to the invention can beconstructed by processes known in the art using chemical synthesis andenzymatic ligation reactions. An antisense nucleic acid (for example anantisense oligonucleotide) can, for example, be synthesized chemically,making use of naturally occurring nucleotides or variously modifiednucleotides which are such that they increase the biological stabilityof the molecules or increase the physical stability of the duplex formedbetween the antisense and the sense nucleic acid; for example,phosphorothioate derivatives and acridine-substituted nucleotides may beused. Examples of modified nucleotides which may be used for generatingthe antisense nucleic acid are, inter alia, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentyladenine, uracil-5-oxyacetic acid (v),wybutoxosin, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)wand 2,6-diaminopurine. The antisense nucleic acid can, alternatively, begenerated biologically using an expression vector into which a nucleicacid has been subcloned in antisense orientation (i.e. RNA which istranscribed by the nucleic acid introduced is in antisense orientationrelative to a target nucleic acid of interest, which is described ingreater detail in the subsection which follows).

The antisense nucleic acid molecules according to the invention areusually administered to a cell or generated in situ so that theyhybridize with, or bind to, the cellular mRNA and/or the genomic DNAencoding a desaturase, thus inhibiting expression of the protein, forexample by inhibiting transcription and/or translation. Hybridizationcan be effected by conventional nucleotide complementarity with theformation of a stable duplex or, for example in the case of an antisensenucleic acid molecule which binds DNA duplices, by specific interactionsin the major groove of the double helix. The antisense molecule can bemodified in such a manner that it specifically binds to a receptor or toan antigen expressed at the selected cell surface, for example bybinding the antisense nucleic acid molecule to a peptide or an antibody,each of which binds to a cell surface receptor or an antigen. The cellscan also be provided with the antisense nucleic acid molecule using thevectors described herein. Vector constructs in which the antisensenucleic acid molecule is under the control of a strong prokaryotic,viral or eukaryotic promoter, including a plant promoter, are preferredfor achieving sufficient intracellular concentrations of the antisensemolecules.

In a further embodiment, the antisense nucleic acid molecule accordingto the invention is an α-anomeric nucleic acid molecule. An a-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA, the strands running parallel to each other, incontrast to ordinary β units (Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641). Moreover, the antisense nucleic acid molecule canencompass a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987)FEBS Lett. 215:327-330).

In a further embodiment, an antisense nucleic acid according to theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which can cleave a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (for example hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used for the catalyticcleavage of desaturase-mRNA transcripts, in order thereby to inhibit thetranslation of desaturase mRNA. A ribozyme with specificity for adesaturase-encoding nucleic acid can be designed on the basis of thenucleotide sequence of one of the desaturase-cDNAs disclosed in SEQ IDNO: 1, 3, 5 or 11 (i.e. or on the basis of a heterologous sequence to beisolated in accordance with the methods taught in the presentinvention). For example, a derivative of a Tetrahymena-L-19-IVS RNA canbe constructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in adesaturase-encoding mRNA. See, for example, Cech et al., U.S. Pat. No.4,987,071 and Cech et al., U.S. Pat. No. 5,116,742. As an alternative,desaturase mRNA can be used for selecting a catalytic RNA with aspecific ribonuclease activity from among a pool of RNA molecules. See,for example, Bartel, D., and Szostak, J. W. (1993) Science261:1411-1418.

As an alternative, desaturase gene expression can be inhibited bydirecting nucleotide sequences which are complementary to the regulatoryregion of a desaturase nucleotide sequence (for example a desaturasepromoter and/or enhancer) in such a way that triple helix structures areformed which inhibit the transcription of a desaturase gene in targetcells. See, in general, Helene, C. (1991) Anticancer Drug Res. 6(6)569-84; Helene, C., et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; andMaher. L. J. (1992) Bioassays 14(12):807-815.

B. Gene Construct (=Nucleic Acid Construct, Nucleic Acid Fragment orExpression Cassette)

The expression cassette according to the invention is to be understoodas meaning the sequences mentioned in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5 or SEQ ID NO: 11 which are the result of the genetic code, and/ortheir functional or nonfunctional derivatives, which were advantageouslylinked functionally to one or more regulatory signals for increasinggene expression and which advantageously control the expression of thecoding sequence in the host cell. These regulatory sequences areintended to make possible the targeted expression of the genes and theprotein expression. Depending on the host organism, this may mean, forexample, that the gene is expressed and/or overexpressed only afterinduction or else that it is expressed and/or overexpressed immediately.For example, these regulatory sequences take the form of sequences towhich inductors or repressors bind, thus regulating the expression ofthe nucleic acid. In addition to these novel regulatory sequences, orinstead of these sequences, the natural regulation of these sequencesmay still be present before the actual structural genes and, ifappropriate, may have been modified genetically, so that naturalregulation was eliminated and the expression of the genes increased.However, the gene construct may also have a simpler structure, that isto say no additional regulatory signals have been inserted before thenucleic acid sequence or its derivatives, and the natural promotertogether with its regulation has not been removed. Instead, the naturalregulatory sequence has been mutated in such a way that regulation nolonger takes place and/or gene expression is increased. These modifiedpromoters may also be arranged by themselves in the form ofpart-sequences (=promoter with parts of the nucleic acid sequencesaccording to the invention) before the natural gene in order to increasethe activity. Moreover, the gene construct may advantageously alsocomprise one or more of what are known as enhancer sequences linkedfunctionally to the promoter, and these make possible an increasedexpression of the nucleic acid. It is also possible to insert additionaladvantageous sequences on the 3′ end of the DNA sequences, such asfurther regulatory elements or terminators. TheΔ5-desaturase/Δ6-desaturase and/or Δ12-desaturase genes may be presentin one or more copies in the expression cassette (=gene construct).

In this context, the regulatory sequences or factors can preferably havea positive effect on, and thus increase, the expression of the genesintroduced, as has been described above. An enhancement of theregulatory elements can advantageously take place at the transcriptionallevel by using strong transcription signals such as promoters and/orenhancers. In addition, however, translation may also be enhanced, forexample by increasing the stability of the mRNA.

A further embodiment of the invention are one or more gene constructscomprising one or more sequences which are defined by SEQ ID NO: 1, 3,5, 7, 9 or 11 and which encode polypeptides in accordance with SEQ IDNO: 2, 4, 6, 8, 10 or 12. SEQ ID NO: 1, 3, 5, 7 and 11 are derived fromdesaturases, while SEQ ID NO: 9 encodes an elongase. Desaturases encodeenzymes which introduce a double bond at the Δ5, Δ6 or Δ12 position, thesubstrate having one, two, three or four double bonds. The sequenceshown in SEQ ID NO: 9 encodes an enzyme activity which elongates a fattyacid by at least two carbon atoms, and the homologs, derivatives oranalogs which are linked functionally to one or more regulatory signals,advantageously for increasing gene expression. Examples of theseregulatory sequences are sequences to which inductors or repressorsbind, thus regulating the expression of the nucleic acid. In addition tothese novel regulatory sequences, the natural regulation of thesesequences may still be present before the actual structural genes and,if appropriate, can have been genetically modified so that the naturalregulation has been eliminated and the expression of the genes has beenincreased.

However, the gene construct may also have a simpler structure, that isto say no additional regulatory signals have been inserted before thesequence SEQ ID NO: 1, 3, 5 or 11 or their homologs and the naturalpromoter with its regulation has not been deleted. Instead, the naturalregulatory sequence has been mutated in such a way that regulation nolonger takes place and gene expression is enhanced. The gene constructmay furthermore advantageously encompass one or more of what are knownas enhancer sequences which are linked functionally to the promoter andwhich make possible increased expression of the nucleic acid sequence.It is also possible additionally to insert advantageous sequences at the3′ end of the DNA sequences, for example further regulatory elements orterminators. The desaturase genes and the elongase gene may be presentin one or more copies in the gene construct. They may be present in onegene construct or more than one gene construct. This gene construct orthe gene constructs can be expressed together in the host organism. Inthis context, the gene construct or the gene constructs can be insertedinto one or more vectors and be present in the cell in free form or elseinserted into the genome. It is advantageous for the insertion offurther genes into organisms if further genes are present in the geneconstruct.

Advantageous regulatory sequences for the novel process exist, forexample, in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac,lpp-lac, lacIq−,T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) or λ-P_(L)promoter and are advantageously used in Gram-negative bacteria. Furtheradvantageous regulatory sequences exist, for example, in theGram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Wardet al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33,nos or in the ubiquitin or phaseolin promoter. Advantageous in thiscontext are also inducible promoters, such as the promoters described inEP-A-0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404(Gatz et al., tetracyclin-inducible), EP-A-0 335 528(abscisic-acid-inducible) or WO 93/21334 (ethanol- orcyclohexenol-inducible). Further suitable plant promoters are thepromoter of cytosolic FBPase or the potato ST-LSI promoter (Stockhaus etal., EMBO J. 8, 1989, 2445), the Glycine max phosphoribosylpyrophosphateamidotransferase promoter (Genbank Accession No. U87999) or thenode-specific promoter described in EP-A-0 249 676. Especiallyadvantageous promoters are those which allow expression in tissues whichare involved in fatty acid biosynthesis. Very especially advantageousare seed-specific promoters such as the USP promoter in accordance withthe embodiment, and also other promoters such as the LEB4 (Baeumlein etal., Plant J., 1992, 2 (2):233-239), DC3 (Thomas, Plant Cell 1996,263:359-368), the phaseolin or the napin promotor. Further especiallyadvantageous promoters are seed-specific promoters which can be used formonocots or dicots which are described in U.S. Pat. No. 5,608,152(oilseed rape napin promoter), WO 98/45461 (Arabidopsis oleosinpromoter), U.S. Pat. No. 5,504,200 (Phaseolus vulgaris phaseolinpromoter), WO 91/13980 (Brassica Bce4 promoter), by Baeumlein et al.,Plant J., 1992, 2 (2):233-239 (LeB4 promoter from a legume), thesepromoters being suitable for dicots. The following promoters aresuitable, for example, for monocots: the barley 1 pt-2 or 1 pt-1promoter (WO 95/15389 and WO 95/23230), the barley Hordein promoter, andother suitable promoters described in WO 99/16890.

In principle, it is possible to use all natural promoters with theirregulatory sequences, such as those mentioned above, for the novelprocess. It is also possible and advantageous additionally to usesynthetic promoters.

As described above, the gene construct can also encompass further geneswhich are to be introduced into the organisms. It is possible andadvantageous to introduce into the host organisms, and to expresstherein, regulatory genes such as genes for inductors, repressors orenzymes which, owing to the enzymatic activity, engage in the regulationof one or more genes of a biosynthetic pathway. These genes can be ofheterologous or homologous origin. Moreover, the nucleic acid constructor gene construct may advantageously comprise further biosynthesis genesof the fatty acid or lipid metabolism or else these genes may be presenton a further, or several further, nucleic acid constructs. Abiosynthesis gene of the fatty acid or lipid metabolism which isadvantageously selected is a gene from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acidsynthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme, A-oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases,allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) ortheir combinations.

For expressing the other genes which are present, gene constructsadvantageously encompass further 3′- and/or 5′-terminal regulatorysequences for enhancing expression, and these are selected for optimalexpression as a function of the host organism chosen and the gene(s).These regulatory sequences, as mentioned above, are intended to makepossible the specific expression of the genes and protein expression.Depending on the host organism, this may mean, for example, that thegene is expressed or overexpressed only after induction, or that it isexpressed and/or overexpressed immediately.

Moreover, the regulatory sequences or regulatory factors can preferablyhave an advantageous effect on the expression of the genes which havebeen introduced, thus enhancing them. In this manner, it is possiblethat the regulatory elements are advantageously enhanced at thetranscriptional level, using strong transcription signals such aspromoters and/or enhancers. However, it is furthermore also possible toenhance translation, for example by improving mRNA stability.

C. Recombinant Expression Vectors and Host Cells

A further aspect of the invention relates to vectors, preferablyexpression vectors, comprising a nucleic acid encoding a desaturasealone (or a part thereof) or a nucleic acid construct described underitem B in which the nucleic acid according to the invention is presentalone or in combination with further biosynthesis genes of the fattyacid or lipid metabolism, such as desaturases or elongases. As used inthe present context, the term “vector” refers to a nucleic acid moleculewhich can transport another nucleic acid to which it is bound. One typeof vector is a “plasmid”, which represents a circular double-strandedDNA loop into which additional DNA segments can be ligated. A furthertype of vector is a viral vector, it being possible for additional DNAsegments to be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they havebeen introduced (for example bacterial vectors with a bacterial originof replication, and episomal mammalian vectors). Other vectors (forexample nonepisomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell and are thus replicatedtogether with the host genome. In addition, certain vectors can governthe expression of genes to which they are linked functionally. Thesevectors are referred to as “expression vectors” herein. Usually,expression vectors which are suitable for recombinant DNA techniques cantake the form of plasmids. In the present description, “plasmid” and“vector” may be used interchangeably since the plasmid is the mostfrequently used form of vector. However, the invention is intended toencompass these other forms of expression vectors, such as viral vectors(for example replication-deficient retroviruses, adenoviruses andadeno-related viruses) which exert similar functions. Furthermore, theterm vector is also intended to encompass other vectors known to theskilled worker, such as phages, viruses such as SV40, CMV, baculovirus,adenovirus, transposons, IS elements, phasmids, phagemids, cosmids,linear or circular DNA.

The recombinant expression vectors according to the invention encompassa nucleic acid according to the invention or a gene construct accordingto the invention in a form which is suitable for expressing the nucleicacid in a host cell, which means that the recombinant expression vectorsencompass one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is/are linked functionallyto the nucleic acid sequence to be expressed. In a recombinantexpression vector “linked functionally” means that the nucleotidesequence of interest is bound to the regulatory sequence(s) in such away that expression of the nucleotide sequence is possible and that theyare bound to each other so that both sequences fulfil the predictedfunction which has been ascribed to the sequence (for example in anin-vitro transcription/translation system or in a host cell, when thevector is introduced into the host cell). The term “regulatory sequence”is intended to encompass promoters, enhancers and other expressioncontrol elements (for example polyadenylation signals). These regulatorysequences are described, for example, in Goeddel: Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990), or see: Gruber and Crosby, in: Methods in Plant MolecularBiology and Biotechnolgy, CRC Press, Boca Raton, Fla., Ed.: Glick andThompson, Chapter 7, 89-108, including the references therein.Regulatory sequences encompass those which control the constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich control the direct expression of the nucleotide sequence only incertain host cells under certain conditions. The skilled worker knowsthat the design of the expression vector may depend on factors such asthe choice of the host cell to be transformed, the extent to which thedesired protein is expressed, and the like. The expression vectorsaccording to the invention can be introduced into host cells in order toproduce proteins or peptides, including fusion proteins or fusionpeptides, which are encoded by the nucleic acids as described herein(for example desaturases, mutant forms of desaturases, fusion proteinsand the like).

The recombinant expression vectors according to the invention can bedesigned for expressing desaturases and elongases in prokaryotic andeukaryotic cells. For example, desaturase genes can be expressed inbacterial cells, such as C. glutamicum, insect cells (using baculovirusexpression vectors), yeast and other fungal cells (see Romanos, M. A.,et al. (1992) “Foreign gene expression in yeast: a review”, Yeast8:423-488; van den Hondel, C. A. M. J. J., et al. (1991) “Heterologousgene expression in filamentous fungi”, in: More Gene Manipulations inFungi, J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press:San Diego; and van den Hondel, C. A. M. J. J., & Punt, P. J. (1991)“Gene transfer systems and vector development for filamentous fungi”,in: Applied Molecular Genetics of Fungi, Peberdy, J. F., et al., Ed.,pp. 1-28, Cambridge University Press: Cambridge), algae (Falciatore etal., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the followingtypes: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, inparticular the species Stylonychia lemnae, using vectors and following atransformation method as described in WO 98/01572, and cells ofmulticelled plants (see Schmidt, R. and Willmitzer, L. (1988) “Highefficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon explants” Plant CellRep.:583-586; Plant Molecular Biology and Biotechnology, C Press, BocaRaton, Fla., Chapter 6/7, pp. 71-119 (1993); F. F. White, B. Jenes etal., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993),128-43; Potrykus, Annu Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),205-225 (and references cited therein)) or mammalian cells. Suitablehost cells are furthermore discussed in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). As an alternative, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

In prokaryotes, proteins are usually expressed with vectors containingconstitutive or inducible promoters which control the expression offusion proteins or nonfusion proteins. Fusion vectors add a series ofamino acids to a protein encoded therein, usually on the amino terminusof the recombinant protein, but also on the C terminus or fused withinsuitable regions in the proteins. These fusion vectors usually havethree tasks: 1) to enhance the expression of recombinant protein; 2) toincrease the solubility of the recombinant protein and 3) to support thepurification of the recombinant protein by acting as ligand in affinitypurification. In the case of fusion expression vectors, a proteolyticcleavage site is frequently introduced at the site where the fusionmoiety and the recombinant protein are linked, so that the recombinantprotein can be separated from the fusion unit after purification of thefusion protein. These enzymes and their corresponding recognitionsequences encompass factor Xa, thrombin and enterokinase.

Typical fusion expression vectors are, inter alia, pGEX (PharmaciaBiotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.), where glutathione S-transferase (GST),maltose-E-binding protein or protein A is fused to the recombinanttarget protein. In one embodiment, the desaturase-encoding sequence iscloned into a pGEX expression vector to generate a vector encoding afusion protein which encompasses, from the N terminus to the C terminus,GST-thrombin cleavage site-X-protein. The fusion protein can be purifiedby affinity chromatography using glutathione-agarose resin. Recombinantdesaturase which is not fused to GST can be obtained by cleaving thefusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsare, inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionof the pTrc vector is based on the transcription by host RNA polymerasefrom a hybrid trp-lac fusion promoter. Target gene expression from thepET 11d vector is based on transcription from a T7-gn10-lac fusionpromoter which is mediated by a coexpressed viral RNA polymerase (T7gn1). This viral polymerase is provided by the host strains BL21 (DE3)or HMS174 (DE3) by a resident λ prophage which harbors a T7 gn1 geneunder the transcriptional control of the lacUV 5 promoter.

Other vectors which are suitable for use in prokaryotic organisms areknown to the skilled worker; these vectors are, for example, in E. colipLG338, pACYC184, the pBR series such as pBR322, the pUC series such aspUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCI, in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214,in Corynebacterium pSA77 or pAJ667. A strategy of maximizing theexpression of recombinant protein is to express the protein in a hostbacterium whose ability to cleave the recombinant proteinproteolytically is disrupted (Gottesman, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). A further strategy is to modify the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector, so that theindividual codons for each amino acid are those which are preferentiallyused in a bacterium selected for expression, such as C. glutamicum, etal. (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Modificationof these nucleic acid sequences according to the invention is carriedout by standard techniques of DNA synthesis.

In a further embodiment, the desaturase expression vector is a yeastexpression vector. Examples of vectors for expression in the yeast S.cerevisiae include pYeDesaturasec1 (Baldari et al. (1987) Embo J.6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88(Schultz et al. (1987) Gene 54:113-123) and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and methods for theconstruction of vectors which are suitable for use in other fungi, suchas the filamentous fungi, include those which are described in detailin: van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi”, in: AppliedMolecular Genetics of fungi, J. F. Peberdy et al., Ed., pp. 1-28,Cambridge University Press: Cambridge, or in: More Gene Manipulations inFungi [J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press:San Diego]. Further suitable yeast vectors are, for example, pAG-1,YEp6, YEp13 or pEMBLYe23.

As an alternative, the desaturases according to the invention can beexpressed in insect cells using baculovirus expression vectors.Baculovirus vectors which are available for expressing proteins incultured insect cells (for example Sf9 cells) include the pAc series(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series(Lucklow and Summers (1989) Virology 170:31-39).

The abovementioned vectors are just a short review of possible suitablevectors. Further plasmids are known to the skilled worker and aredescribed, for example, in: Cloning Vectors (Ed. Pouwels, P. H., et al.,Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In yet a further embodiment, a nucleic acid according to the inventionis expressed in mammalian cells using a mammalian expression vector.Mammals for the purposes of the invention are to be understood as allnon-human mammals. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195). When used in mammalian cells, the control functionsof the expression vector are frequently provided by viral regulatoryelements. Promoters which are usually used are derived, for example,from polyoma, adenovirus2, cytomegalovirus and Simian Virus 40. Othersuitable expression systems for prokaryotic and eukaryotic cells can befound in Chapters 16 and 17 of Sambrook, J., Fritsch, E. F., andManiatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector cancontrol the expression of the nucleic acid preferably in a specific celltype (for example, tissue-specific regulatory elements are used forexpressing the nucleic acid). Tissue-specific regulatory elements areknown in the art. Nonlimiting examples of suitable tissue-specificpromoters are, inter alia, the albumen promoter (liver-specific; Pinkertet al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calameand Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters ofT-cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (forexample neurofilament promoter; Byrne and Ruddle (1989) PNAS86:5473-5477), pancreas-specific promoters (Edlund et al., (1985)Science 230:912-916) and mamma-specific promoters (for example milkserum promoter; U.S. Pat. No. 4,873,316 and European Patent Applicationdocument No. 264,166). Also included are development-regulatedpromoters, for example the mouse hox promoters (Kessel and Gruss (1990)Science 249:374-379) and the fetoprotein promoter (Campes and Tilghman(1989) Genes Dev. 3:537-546).

In a further embodiment, the desaturases according to the invention canbe expressed in single-celled plant cells (such as algae), seeFalciatore et al., 1999, Marine Biotechnology 1 (3):239-251 andreferences cited therein, and plant cells from higher plants (forexample spermatophytes such as crops). Examples of plant expressionvectors include those which are described in detail in: Becker, D.,Kemper, E., Schell, J., and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press, 1993, pp. 15-38.

A plant expression cassette preferably comprises regulatory sequenceswhich can control gene expression in plant cells and which are linkedfunctionally so that each sequence can fulfil its function, such astranscriptional termination, for example polyadenylation signals.Preferred polyadenylation signals are those derived from Agrobacteriumtumefaciens T-DNA, such as gene 3 of the Ti plasmid pTiACH5, which isknown as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.)or functional equivalents thereof, but all other terminators which arefunctionally active in plants are also suitable.

Since plant gene expression is very frequently not limited to thetranscription level, a plant expression cassette preferably comprisesother functionally linked sequences, such as translation enhancers, forexample the overdrive sequence, which contains the 5′-untranslatedtobacco mosaic virus leader sequence, which increases the protein/RNAratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).

Plant gene expression must be linked functionally to a suitable promoterwhich effects gene expression in a cell- or tissue-specific manner withthe correct timing. Preferred promoters are those which lead toconstitutive expression (Benfey et al., EMBO J. 8 (1989) 2195-2202),such as those which are derived from plant viruses such as 35S CAMV(Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat.No. 5,352,605 and WO 84/02913) or plant promoters such as the Rubiscosmall subunit promoter described in U.S. Pat. No. 4,962,028.

Other sequences which are preferred for use for functional linkage inplant gene expression cassettes are targeting sequences which arerequired for targeting the gene product into its corresponding cellcompartment (for a review, see Kermode, Crit. Rev. Plant Sci. 15, 4(1996) 285-423 and references cited therein), for example into thevacuole, the nucleus, all types of plastids such as amyloplasts,chloroplasts, chromoplasts, the extracellular space, the mitochondria,the endoplasmic reticulum, elaioplasts, peroxisomes and othercompartments of plant cells.

Plant gene expression can also be facilitated via a chemically induciblepromoter (for a review, see Gatz 1997, Annu. Rev. Plant Physiol. PlantMol. Biol., 48:89-108). Chemically inducible promoters are particularlysuitable when it is desired for gene expression to take place in aspecific manner with regard to timing. Examples of such promoters are asalicylic acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter.

Other suitable promoters are promoters which respond to biotic orabiotic stress conditions, for example the pathogen-induced PRP1 genepromoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), theheat-inducible tomato hsp80 promoter (U.S. Pat. No. 5,187,267), the lowtemperature-inducible potato alpha-amylase promoter (WO 96/12814) or thewound-inducible pinII promoter (EP-A-0 375 091).

Promoters which are particularly preferred are those which lead to geneexpression in tissues and organs in which lipid and oil biosynthesistake place, in seed cells such as endosperm cells and cells of thedeveloping embryo. Promoters which are suitable are the oilseed rapenapin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992,Plant Journal, 2 (2):233-9), and promoters which lead to theseed-specific expression in monocots such as maize, barley, wheat, rye,rice and the like. Notable promoters which are suitable are the barleylpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene).

The multiparallel expression of desaturases according to the invention,alone or in combination with other desaturases or elongases, may bedesired in particular. The introduction of such expression cassettes canbe effected by a simultaneous transformation of a plurality ofindividual expression constructs or by combining a plurality ofexpression cassettes on one construct. Also, a plurality of vectors canbe transformed with in each case a plurality of expression cassettes,and transferred to the host cell.

Promoters which are also particularly suitable are those which lead toplastid-specific expression, since plastids are the compartment in whichthe precursors and some end products of lipid biosynthesis aresynthesized. Suitable promoters such as the viral RNA polymerasepromoter are described in WO 95/16783 and WO 97/06250, and theArabidopsis clpP promoter, described in WO 99/46394.

The invention furthermore provides a recombinant expression vectorencompassing a DNA molecule according to the invention which is clonedinto the expression vector in antisense orientation, i.e. the DNAmolecule is linked functionally to a regulatory sequence in such a waythat it allows the expression (by transcribing the DNA molecule) of anRNA molecule which is “antisense” to the desaturase mRNA. Regulatorysequences may be selected which are linked functionally to a nucleicacid cloned in antisense orientation and which control the continuousexpression of the antisense RNA molecule in a multiplicity of celltypes, for example, viral promoters and/or enhancers or regulatorysequences may be selected which control the constitutive,tissue-specific or cell-type-specific expression of antisense RNA. Theantisense expression vector may be present in the form of a recombinantplasmid, phagemid or attenuated virus in which the antisense nucleicacids are produced under the control of a highly effective regulatoryregion whose activity can be determined by the cell type into which thevector has been introduced. For an explanation of the regulation of geneexpression by means of antisense genes, see Weintraub, H., et al.,Antisense-RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986.

A further aspect of the invention relates to host cells into which arecombinant expression vector according to the invention has beenintroduced. The terms “host cell” and “recombinant host cell” are usedinterchangeably in the present context. Naturally, these terms do notonly refer to the particular target cell, but also to the progeny orpotential progeny of this cell. Since specific modifications may occurin subsequent generations owing to mutation or environmental effects,this progeny is not necessarily identical with the parental cell, butremains within the scope of the term as used in the present context.

The terms recombinant or transgene, for example recombinant expressionvector or recombinant host or host cells is to be understood as meaning,for the purpose of the invention, that the nucleic acids according tothe invention and/or their natural regulatory sequences at the 5′ and 3′positions of the nucleic acids are not in their natural environment,that is to say either the location of the sequences in the originalorganism was altered or the nucleic acid sequences and/or the regulatorysequences were mutated in it or the nucleic acid sequences according tothe invention were transferred into an organism other than the originalorganism or their regulatory sequences. Combinations of thesemodifications are also possible. Natural environment is to be understoodas meaning the location of a nucleic acid sequence in an organism as itoccurs in nature.

A host cell may be a prokaryotic or eukaryotic cell. For example adesaturase can be expressed in bacterial cells such as C. glutamicum,insect cells, fungal cells or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi orother microorganisms such as C. glutamicum. Other suitable host cellsare known to the skilled worker.

Vector DNA can be introduced into prokaryotic or eukaryotic cells byconventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction asused in the present context are intended to encompass a multiplicity ofmethods known in the art for introducing foreign nucleic acid (forexample DNA) into a host cell, including calcium phosphate or calciumchloride coprecipitation, DEAE-dextran-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory text books,such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

It is known about the stable transfection of mammalian cells that only asmall number of the cells integrate the foreign DNA into their genome,depending on the expression vector used and the transfection techniqueused. To identify and select these integrants, a gene which encodes aselectable marker (for example resistance to antibiotics) is usuallyintroduced into the host cells together with the gene of interest.Preferred selectable markers encompass those which impart resistance todrugs such as G418, hygromycin and methotrexate, or, in plants, thosewhich impart resistance to a herbicide such as glyphosate orglufosinate. Further suitable markers are, for example, markers whichencode genes which are involved in the biosynthetic pathways of, forexample, sugars or amino acids, such as β-galactosidase, ura3 or ilv2.Markers which encode genes such as luciferase, gfp or other fluorescencegenes are also suitable. These markers can be used in mutants in whichthese genes are not functional since they have been deleted for exampleby means of conventional methods. Furthermore, markers which encode anucleic acid which encodes a selectable marker can be introduced into ahost cell on the same vector as the one which encodes a desaturase, orcan be introduced on a separate vector. Cells which have beentransfected stably with the nucleic acid introduced can be identifiedfor example by drug selection (for example, cells which have theselectable marker integrated survive, whereas the other cells die).

To generate a microorganism with homologous recombination, a vector isgenerated which contains at least one segment of a desaturase gene intowhich a deletion, addition or substitution has been introduced in orderto modify the desaturase gene hereby, for example to functionallydisrupt it. This desaturase gene is preferably a Phaeodactylumtricornutum desaturase gene, but a homolog or analog from otherorganisms, even from mammalian, fungal or insect cells, can also beused. In a preferred embodiment, the vector is designed in such a waythat the endogenous desaturase gene is functionally disrupted (i.e. nolonger encodes a functional protein, also termed knock-out vector) uponhomologous recombination. As an alternative, the vector can be designedin such a way that the endogenous desaturase gene is mutated or modifiedotherwise upon homologous recombination while still encoding afunctional protein (for example, the upstream regulatory region can bemodified in such a way that this leads to a modification of theexpression of the endogenous desaturase). To generate a point mutationvia homologous recombination, DNA-RNA hybrids, which are also known aschimeraplasty, and which are known from Cole-Strauss et al., 1999,Nucleic Acids Research 27(5):1323-1330 and Kmiec, Gene therapy, 1999,American Scientist, 87(3):240-247 can also be used.

In the vector for homologous recombination, the modified segment of thedesaturase gene is flanked at its 5′ and 3′ end by additional nucleicacid of the desaturase gene, so that homologous recombination ispossible between the exogenous desaturase gene which is present on thevector and an endogenous desaturase gene in a microorganism or plant.The additional flanking desaturase nucleic acid is sufficiently long forsuccessful homologous recombination with the endogenous gene. Usually,several hundred base pairs up to kilobases of flanking DNA (both on the5′ and on the 3′ end) are present in the vector (for a description ofvectors for homologous recombination, see, for example, Thomas, K. R.,and Capecchi, M. R. (1987) Cell 51:503 or for the recombination inPhyscomitrella patens on cDNA basis, see Strepp et al., 1998, Proc.Natl. Acad. Sci. USA 95 (8):4368-4373). The vector is introduced into amicroorganism or plant cell (for example by means of polyethyleneglycol-mediated DNA), and cells in which the desaturase gene introducedhas undergone homologous recombination with the endogenous desaturasegene are selected using techniques known in the art.

In another embodiment, recombinant organisms such as microorganisms canbe generated which contain selected systems which allow regulatedexpression of the gene introduced. The inclusion of a desaturase gene ina vector, where it is placed under the control of the lac operon,allows, for example, expression of the desaturase gene only in thepresence of IPTG. These regulatory systems are known in the art.

A host cell according to the invention, such as a prokaryotic oreukaryotic host cell, growing either in culture or in a field, can beused for producing (i.e. expressing) a desaturase. In plants, analternative method can additionally be used by directly transferring DNAinto developing flowers via electroporation or Agrobacterium-mediatedgene transfer. Accordingly, the invention furthermore provides methodsof producing desaturases using the host cells according to theinvention. In one embodiment, the method encompasses growing the hostcell according to the invention (into which a recombinant expressionvector encoding a desaturase has been introduced or into whose genome agene encoding a wild-type or modified desaturase has been introduced) ina suitable medium until the desaturase has been produced. In a furtherembodiment, the method encompasses isolating the desaturases from themedium or the host cell.

Host cells which are suitable in principle for taking up the nucleicacid according to the invention, the gene product according to theinvention or the vector according to the invention are all prokaryoticor eukaryotic organisms. The host organisms which are usedadvantageously are organisms such as bacteria, fungi, yeasts, animalcells or plant cells. Further advantageous organisms are animals or,preferably, plants or parts thereof. Fungi, yeasts or plants arepreferably used, especially preferably fungi or plants, very especiallypreferably plants such as oil crop plants which contain large amounts oflipid compounds, such as oilseed rape, evening primrose, canola, peanut,linseed, soybean, safflower, sunflower, borage, or plants such as maize,wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper,tagetes, Solanaceae plants such as potato, tobacco, egg-plant andtomato, Vicia species, pea, alfalfa, bush plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut) and perennial grasses andfodder crops. Especially preferred plants according to the invention areoil crop plants such as soybean, peanut, oilseed rape, canola, linseed,evening primrose, sunflower, safflower, trees (oil palm, coconut).

D. Isolated Desaturase

A further aspect of the invention relates to isolated desaturases andbiologically active parts thereof. An “isolated” or “purified” proteinor a biologically active part thereof is essentially free of cellularmaterial when it is produced by recombinant DNA techniques, or free fromchemical precursors or other chemicals when it is synthesizedchemically. The term “essentially free of cellular material” encompassesdesaturase preparations in which the protein is separated from cellularcomponents of the cells in which it is produced naturally orrecombinantly. In one embodiment, the term “essentially free of cellularmaterial” encompasses desaturase preparations with less thanapproximately 30% (based on the dry weight) of non-desaturase (alsoreferred to herein as “contaminating protein”), more preferably lessthan approximately 20% of non-desaturase, even more preferably less thanapproximately 10% of non-desaturase and most preferably less thanapproximately 5% of non-desaturase. If the desaturase or a biologicallyactive part thereof has been produced recombinantly, it is alsoessentially free of culture medium, i.e. the culture medium amounts toless than approximately 20%, more preferably less than approximately 10%and most preferably less than approximately 5% of the volume of theprotein preparation. The term “essentially free from chemical precursorsor other chemicals” encompasses desaturase preparations in which theprotein is separate from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, theterm “essentially free of chemical precursors or other chemicals”encompasses desaturase preparations with less than approximately 30%(based on the dry weight) of chemical precursors or non-desaturasechemicals, more preferably less than approximately 20% of chemicalprecursors or non-desaturase chemicals, even more preferably less thanapproximately 10% of chemical precursors or non-desaturase chemicals andmost preferably less than approximately 5% of chemical precursors ornon-desaturase chemicals. In preferred embodiments, isolated proteins orbiologically active parts thereof exhibit no contaminating proteins fromthe same organisms from which the desaturase originates. These proteinsare usually produced by recombinant expression, for example,Phaeodactylum tricornutum desaturase in plants such as Physcomitrellapatens or abovementioned microorganisms, for example bacteria such as E.coli, Bacillus subtilis, C. glutamicum, fungi such as Mortierella,yeasts such as Saccharomyces, or ciliates such as Colpidium or algaesuch as Phaeodactylum.

An isolated desaturase according to the invention or a part thereof canalso participate in the metabolism of compounds required for thesynthesis of cell membranes in Phaeodactylum tricornutum or in thetransport of molecules via these membranes. In preferred embodiments,the protein or the part thereof encompasses an amino acid sequence whichhas sufficient homology with an amino acid sequence of SEQ ID NO: 2, 4,6 or 12 for the protein or part thereof to retain the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes in Phaeodactylum tricornutum or in the transport ofmolecules via these membranes. The part of the protein is preferably abiologically active part as described herein. In a further preferredembodiment, a desaturase according to the invention has one of the aminoacid sequences shown in SEQ ID NO: 2, 4, 6 or 12. In a further preferredembodiment, the desaturase has an amino acid sequence which is encodedby a nucleotide sequence which hybridizes with a nucleotide sequence ofSEQ ID NO: 1, 3, 5 or 11, for example under stringent conditions. In yetanother preferred embodiment, the desaturase has an amino acid sequencewhich is encoded by a nucleotide sequence which has at leastapproximately 50 to 60%, preferably at least approximately 60 to 70%,more preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%,and even more preferably at least approximately 96%, 97%, 98%, 99% ormore homology with one of the amino acid sequences of SEQ ID NO: 2, 4, 6or 18. The desaturase preferred according to the invention preferablyalso has at least one of the desaturase activities described herein. Forexample, a desaturase preferred according to the invention encompassesan amino acid sequence encoded by a nucleotide sequence which hybridizeswith a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, for exampleunder stringent conditions, and which can participate in the metabolismof compounds required for the synthesis of cell membranes inPhaeodactylum tricornutum or in the transport of molecules via thesemembranes and is capable of introducing a double bond into a fatty acidwith one, two, three or four double bonds and a chain length of C₁₈, C₂₀or C₂₂.

In other embodiments, the desaturase is essentially homologous with anamino acid sequence of SEQ ID NO: 2, 4 or 6 and retains the functionalactivity of the protein of one of the sequences of SEQ ID NO: 2, 4 or 6,the amino acid sequence differing, however, owing to natural variationor mutagenesis as described in detail in the above subsection I. In afurther embodiment, the desaturase is, accordingly, a proteinencompassing an amino acid sequence which has at least approximately 50to 60% homology, preferably approximately 60 to 70% homology and morepreferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%homology and most preferably at least approximately 96%, 97%, 98%, 99%or more homology with a complete amino acid sequence of SEQ ID NO: 2, 4or 6 and has at least one of the desaturase activities described herein.In another embodiment, the invention relates to a complete Phaeodactylumtricornutum protein which is essentially homologous with a completeamino acid sequence of SEQ ID NO: 2, 4 or 6.

Biologically active parts of a desaturase encompass peptidesencompassing amino acid sequences derived from the amino acid sequenceof a desaturase, for example an amino acid sequence shown in SEQ ID NO:2, 4 or 6 or the amino acid sequence of a protein which is homologouswith a desaturase, which peptides have fewer amino acids than thefull-length desaturase or the full-length protein which is homologouswith a desaturase and have at least one activity of a desaturase.Biologically active parts (peptides, for example peptides with a lengthof, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 ormore amino acids) usually encompass a domain or a motif with at leastone activity of a desaturase. Moreover, other biologically active partsin which other regions of the protein are deleted can be generated byrecombinant techniques and examined for one or more of the activitiesdescribed herein. The biologically active parts of the desaturasepreferably encompass one or more selected domains/motifs or partsthereof with biological activity.

Desaturases are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above), and the desaturase isexpressed in the host cell. The desaturase can then be isolated from thecells by a suitable purification scheme using standard techniques ofprotein purification. As an alternative to the recombinant expression, adesaturase, a desaturase polypeptide or a desaturase peptide can besynthesized chemically by means of standard techniques of peptidesynthesis. Moreover, native desaturase can be isolated from cells (forexample endotheliol cells), for example using an anti-desaturaseantibody which can be raised by standard techniques, using a desaturaseaccording to the invention or a fragment thereof

The invention also provides chimeric desaturase proteins or desaturasefusion proteins. As used in the present context, a “chimeric desaturaseprotein” or “desaturase fusion protein” encompasses a desaturasepolypeptide which is bound functionally to a non-desaturase polypeptide.A “desaturase polypeptide” refers to a polypeptide with an amino acidsequence which corresponds to a desaturase, whereas a “non-desaturasepolypeptide” refers to a polypeptide with an amino acid sequence whichcorresponds to a protein which is essentially not homologous with thedesaturase, for example a protein which differs from the desaturase andwhich originates from the same or another organism. Within the fusionprotein, the term “linked functionally” is understood as meaning thatthe desaturase polypeptide and the non-desaturase polypeptide are fusedto each other in such a way that both sequences fulfil the predictedfunction which has been ascribed to the sequence used. Thenon-desaturase polypeptide can be fused to the N terminus or the Cterminus of the desaturase polypeptide. In one embodiment, the fusionprotein is, for example, an EST-desaturase fusion protein in which thedesaturase sequences are fused to the C terminus of the GST sequences.These fusion proteins can facilitate the purification of the recombinantdesaturases. In a further embodiment, the fusion protein is a desaturasewhich has a heterologous signal sequence (N terminus). In specific hostcells (for example mammalian host cells), the expression and/orsecretion of a desaturase can be increased by using a heterologoussignal sequence.

A chimeric desaturase protein or desaturase fusion protein according tothe invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments which encode different polypeptide sequences areligated to each other in-frame using conventional techniques, forexample by employing blunt ends or overhanging ends for ligation,restriction enzyme cleavage for providing suitable ends, filling upcohesive ends, as required, treatment with alkaline phosphatase to avoidundesired linkages, and enzymation ligation. In a further embodiment,the fusion gene can be synthesized by conventional techniques includingDNA synthesizers. As an alternative, PCR amplification of gene fragmentscan be carried out using anchor primers which generate complementaryoverhangs between successive gene fragments which can subsequently behybridized with each other and reamplified to give rise to a chimericgene sequence (see, for example, Current Protocols in Molecular Biology,Ed. Ausubel et al., John Wiley & Sons: 1992). Moreover, a large numberof expression vectors which already encode a fusion unit (for example aGST polypeptide) are commercially available. A desaturase-encodingnucleic acid can be cloned into such an expression vector so that thefusion unit is linked in-frame to the desaturase protein.

Desaturase homologs can be generated by mutagenesis, for example byspecific point mutation or by truncating the desaturase. The term“homologs” as used in the present context refers to a variant form ofthe desaturase which acts as agonist or antagonist with the desaturaseactivity. A desaturase agonist can essentially retain the same activityas the desaturase, or some of the biological activities of thedesaturase. A desaturase antagonist can inhibit one or more activitiesof the naturally occurring desaturase form, for example by competitivebinding to an upstream or downstream element of the metabolic cascadefor cell membrane components which encompass the desaturase, or bybinding to a desaturase which mediates the transport of compounds viacell membranes, thus inhibiting translocation.

In an alternative embodiment, desaturase homologs can be identified byscreening combinatory libraries of desaturase mutants, for exampletruncated mutants, with regard to desaturase agonist or antagonistactivity. In one embodiment, a variegated library of desaturase variantsis generated at nucleic acid level by combinatory mutagenesis andencoded by a variegated genetic library. A variegated library ofdesaturase variants can be generated for example by enzymatic ligationof a mixture of synthetic oligonucleotides into gene sequences so that adegenerate set of potential desaturase sequences can be expressed asindividual polypeptides or, alternatively, as a set of larger fusionproteins (for example for phage display) which comprise this set ofdesaturase sequences. There is a multiplicity of methods which can beused for generating libraries of potential desaturase homologs from adegenerate oligonucleotide sequence. The chemical synthesis of adegenerate gene sequence can be carried out in a DNA synthesizer, andthe synthetic gene can then be ligated into a suitable expressionvector. The use of the degenerate set of genes allows all sequenceswhich encode the desired set of potential desaturase sequences to beprovided in a mixture. Methods for the synthesis of degenerateoligonucleotides are known in the art (see, for example, Narang, S. A.(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev. Biochem.53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983)Nucleic Acids Res. 11:477).

In addition, libraries of desaturase fragments can be used forgenerating a variegated population of desaturase fragments for screeningand for the subsequent selection of homologs of a desaturase. In oneembodiment, a library of fragments of the coding sequence can begenerated by treating a double-strand PCR fragment of a codingdesaturase sequence with a nuclease under conditions under whichdouble-strand breaks only occur approximately once per molecule,denaturing the double-stranded DNA, renaturing the DNA with theformation of double-stranded DNA which can encompass sense/antisensepairs of various products with double-strand breaks, removal ofsingle-stranded sections from newly formed duplices by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. Using this method, an expression library can be derived whichencodes N-terminal, C-terminal and internal desaturase fragments ofvarious sizes.

A number of techniques for screening gene products in combinatorylibraries which have been generated by point mutation or truncation andfor screening cDNA libraries for gene products with a selected propertyare known in the art. These techniques can be adapted to rapid screeningof the gene libraries which have been generated by combinatorymutagenesis of desaturase homologs. The most frequently used techniquesfor screening large gene libraries which can be subjected tohigh-throughput analysis usually encompass cloning the gene library intoreplicable expression vectors, transforming suitable cells with theresulting vector library, and expressing the combinatory genes underconditions under which detecting the desired activity facilitates theisolation of the vector encoding the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a novel technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening assays for identifying desaturasehomologs (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

A further known technique for modifying catalytic properties of enzymesor the genes encoding them is gene shuffling (see, for example, Stemmer,PNAS 1994, 91: 10747-10751, WO 97/20078 or WO 98/13487), which is acombination of gene fragments where this new combination canadditionally be varied by erroneous polymerase chain reactions thuscreating a high sequence diversity to be assayed. However, theprerequisite for using such an approach is a suitable screening systemfor testing the resulting gene diversity for functionality.

A screening method which identifies a PUFA-dependent enzyme activity oractivities, is a prerequisite in particular for screening desaturaseactivities. As regards desaturase activities with a specificity forPUFAs, the toxicity of arachidonic acid in the presence of a toxicmetabolyte (here: salicylic acid or salicylic acid derivatives) can beexploited in Mucor species which can be transformed with desired geneconstructs by known transformation methods (Eroshin et al.,Mikrobiologiya, Vol. 65, No.1 1996, pages 31-36), to carry out agrowth-based primary screening. Resulting clones can then be analyzedfor their lipid constituents by means of gas chromatography and massspectroscopy in order to identify the nature and quantity of startingmaterials and products.

In a further embodiment, cell-based assays can be made use of foranalyzing a variegated desaturase library using further processes knownin the art.

E. Uses and Processes/Methods According to the Invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors and host cells described herein can be used in one ormore of the processes/methods which follow: identification ofPhaeodactylum and related organisms, genome mapping of organisms whichare related to Phaeodactylum tricornutum, identification andlocalization of Phaeodactylum tricornutum sequences of interest,evolutionary studies, determination of desaturase protein regionsrequired for the function, modulation of a desaturase activity,modulation of the metabolism of one or more cell membrane components,modulation of the transmembrane transport of one or more compounds, andmodulation of the cellular production of a desired compound such as afine chemical. The desaturase nucleic acid molecules according to theinvention have a multiplicity of uses. Firstly, they can be used foridentifying an organism as Phaeodactylum tricornutum or a close relativethereof. They can also be used for identifying the presence ofPhaeodactylum tricornutum or of a relative thereof in a mixed populationof microorganisms. The invention provides the nucleic acid sequences ofa series of Phaeodactylum tricornutum genes; the presence or absence ofthis organism can be determined by screening the extracted genomic DNAof a culture of a uniform or mixed population of microorganisms understringent conditions with a probe covering a region of a Phaeodactylumtricornutum gene or parts thereof, which gene is unique to thisorganism. Phaeodactylum tricornutum itself is used for the commercialproduction of polyunsaturated acids and is additionally suitable for theproduction of PUFAs, also in other organisms, in particular when it isintended for the resulting PUFAs also to be incorporated into thetriacylglycerol fraction.

Furthermore, the nucleic acid and protein molecules according to theinvention can act as markers for specific regions of the genome. This issuitable not only for mapping the genome, but also for functionalPhaeodactylum tricornutum proteins. To identify the genome region towhich a certain DNA-binding protein of Phaeodactylum tricornutum binds,it might be possible, for example, to fragment the Phaeodactylumtricornutum genome, and the fragments could be incubated with theDNA-binding protein. Those which bind the protein can additionally bescreened with the nucleic acid molecules according to the invention,preferably with readily detectable markers; the binding of such anucleic acid molecule to the genome fragment makes possible thelocalization of the fragment on the genome map of Phaeodactylumtricornutum and, if this is carried out repeatedly with differentenzymes, facilitates a rapid determination of the nucleic acid sequenceto which the protein binds. Moreover, the nucleic acid moleculesaccording to the invention can have sufficient homology with thesequences of related species for these nucleic acid molecules to be ableto act as markers for the construction of a genomic map in related fungior algae.

The desaturase nucleic acid molecules according to the invention arealso suitable for evolutionary studies and studies of the proteinstructure. The metabolic and transport processes in which the moleculesaccording to the invention are involved are utilized by many prokaryoticand eukaryotic cells; the evolutionary degree of relatedness of theorganisms can be determined by comparing the sequences of the nucleicacid molecules according to the invention with those which encodesimilar enzymes from other organisms. Accordingly, such a comparisonallows the determination of which sequence regions are conserved andwhich are not conserved, and this may be helpful when determiningregions of the protein which are essential for enzyme function. Thistype of determination is valuable for protein engineering studies andmay provide a clue of how much mutagenesis the protein can toleratewithout losing its function.

Manipulation of the desaturase nucleic acid molecules according to theinvention can lead to the production of desaturases with functionaldifferences to the wild-type desaturases. The efficiency or activity ofthese proteins can be improved, they can be present in the cell inlarger numbers than usual, or their efficiency or activity can bereduced. An improved efficiency or activity means, for example, that theenzyme has a higher selectivity and/or activity, preferably an activitywhich is at least 10% higher, especially preferably an activity which isat least 20% higher, very especially preferably an activity which is atleast 30% higher than that of the original enzyme.

There exists a series of mechanisms by which modification of adesaturase according to the invention can directly affect the yield,production and/or production efficiency of a fine chemical comprisingsuch a modified protein. Obtaining fine chemical compounds from culturesof ciliates, algae or fungi on a large scale is significantly improvedwhen the cell secretes the desired compounds, since these compounds canbe isolated readily from the culture medium (in contrast to extractionfrom the biomass of the cultured cells). Otherwise, purification can beimproved when the cell stores compounds in-vivo in a specializedcompartment with a sort of concentration mechanism. In plants whichexpress desaturases, an increased transport may lead to betterdistribution within the plant tissue and the plant organs. Increasingthe number or the activity of transporter molecules which export finechemicals from the cell may allow the quantity of the fine chemicalsproduced, which is present in the extracellular medium, to be increased,thus facilitating harvesting and purification or, in the case of plants,more efficient distribution. In contrast, increased amounts ofcofactors, precursor molecules and intermediates for the suitablebiosynthetic pathways are required for efficient overproduction of oneor more fine chemicals. Increasing the number and/or the activity oftransporter proteins involved in the import of nutrients such as carbonsources (i.e. sugars), nitrogen sources (i.e. amino acids, ammoniumsalts), phosphate and sulfur can improve the production of a finechemical owing to the elimination of all limitations of the nutrientsavailable in the biosynthetic process. Fatty acids such as PUFAs andlipids comprising PUFAs are desirable fine chemicals themselves;optimizing the activity or increasing the number of one or moredesaturases according to the invention involved in the biosynthesis ofthese compounds, or disrupting the activity of one or more desaturasesinvolved in the catabolism of these compounds, can thus increase theyield, production and/or production efficiency of fatty acids and lipidmolecules in ciliates, algae, plants, fungi, yeasts or othermicroorganisms.

The manipulation of one or more desaturase genes according to theinvention can likewise lead to desaturases with modified activitieswhich indirectly affect the production of one or more desired finechemicals from algae, plants, ciliates or fungi. The normal biochemicalmetabolic processes leaked, for example, to the production of amultiplicity of waste products (for example hydrogen peroxide and otherreactive oxygen species) which can actively disrupt these metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thus inactivating some enzymes with tyrosin in the active center(Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3(2);226-235)). Whilethese waste products are normally excreted, the cells used forfermentative production on a large scale are optimized for theoverproduction of one or more fine chemicals and can therefore producemore waste products than is customary for a wild-type cell. Optimizingthe activity of one or more desaturases according to the inventioninvolved in the export of waste molecules allows the improvement of theviability of the cell and the maintenance of an efficient metabolicactivity. Also, the presence of high intracellular amounts of thedesired fine chemical can in fact be toxic to the cell, so that theviability of the cell can be improved by increasing the ability of thecell to secrete these compounds.

Furthermore, the desaturases according to the invention can bemanipulated in such a way that the relative amounts of various lipidsand fatty acid molecules are modified. This can have a decisive effecton the lipid composition of the cell membrane. Since each lipid type hasdifferent physical properties, a modification of the lipid compositionof the membrane can significantly modify membrane fluidity. Changes inmembrane fluidity can affect the transport of molecules via the membranewhich, as explained above, can modify the export of waste products or ofthe fine chemical produced or the import of nutrients which arerequired. These changes in membrane fluidity can also have a decisiveeffect on cell integrity; cells with comparatively weaker membranes aremore susceptible to abiotic and biotic stress conditions which candamage or kill the cell. Manipulation of desaturases involved in theproduction of fatty acids and lipids for membrane synthesis so that theresulting membrane has a membrane composition which is more susceptibleto the environmental conditions prevailing in the cultures used for theproduction of fine chemicals should allow more cells to survive andmultiply. Larger numbers of producing cells should manifest themselvesin greater yields, higher production or higher production efficiency ofthe fine chemical from the culture.

The abovementioned mutagenesis strategies for desaturases intended tolead to elevated yields of a fine chemical are not to be construed aslimiting; variations of these strategies are readily obvious to theskilled worker. Using these mechanisms, and with the aid of themechanisms disclosed herein, the nucleic acid and protein moleculesaccording to the invention can be used for generating algae, ciliates,plants, animals, fungi or other microorganisms such as C. glutamicum,which express mutated desaturase nucleic acid and protein molecules sothat the yield, production and/or production efficiency of a desiredcompound is improved. This desired compound can be any natural productof algae, ciliates, plants, animals, fungi or bacteria which encompassesthe end products of biosynthetic pathways and intermediates of naturallyoccurring metabolic pathways, and also molecules which do not naturallyoccur in the metabolism of these cells, but which are produced by thecells according to the invention.

A further embodiment according to the invention is a process for theproduction of PUFAs, which comprises culturing an organism whichcontains a nucleic acid according to the invention, a gene constructaccording to the invention or a vector according to the invention whichencode a polypeptide which elongates C₁₈-, C₂₀- or C₂₂-fatty acids withat least two double bonds in the fatty acid molecule by at least twocarbon atoms under conditions under which PUFAs are produced in theorganism. PUFAs produced by this process can be isolated by harvestingthe organisms either from the culture in which they grow or from thefield, and disrupting and/or extracting the harvested material with anorganic solvent. The oil, which contains lipids, phospholipids,sphingolipids, glycolipids, triacylglycerols and/or free fatty acidswith a higher PUFA content can be isolated from this solvent. The freefatty acids with a higher PUFA content can be isolated by basic or acidhydrolysis of the lipids, phospholipids, sphingolipids, glycolipids andtriacylglycerols. A higher PUFA content means at least 5%, preferably10%, especially preferably 20%, very especially preferably 40% morePUFAs than the original organism which does not have additional nucleicacid encoding the desaturase according to the invention.

The PUFAs produced by this process are preferably C₁₈- or C₂₀₋₂₂-fattyacid molecules with at least two double bonds in the fatty acidmolecule, preferably three, four, in combination with a further elongaseand a Δ4-desaturase five or six double bonds. These C₁₈- or C₂₀₋₂₂-fattyacid molecules can be isolated from the organism in the form of an oil,lipid or a free fatty acid. Examples of suitable organisms are thosementioned above. Preferred organisms are transgenic plants.

An embodiment according to the invention are oils, lipids or fatty acidsor fractions thereof which have been prepared by the above-describedprocess, especially preferably an oil, a lipid or a fatty acidcomposition comprising PUFAs and originating from transgenic plants.

A further embodiment according to the invention is the use of the oil,lipid or fatty acid composition in feeds, foods, cosmetics orpharmaceuticals.

The invention further relates to a method of identifying an antagonistor agonist of desaturases, comprising

-   a) contacting the cells which express the polypeptide of the present    invention with a candidate substance;-   b) testing the desaturate activity;-   c) comparing the desaturase activity with a standard activity in the    absence of the candidate material, where an increase in the    desaturase activity beyond the standard indicates that the candidate    material is an agonist and a reduction in the desaturase activity    indicates that the candidate material is an antagonist.

The candidate substance mentioned can be a substance which has beensynthesized chemically or produced by microbes and can occur, forexample, in cell extracts of, for example, plants, animals ormicroorganisms. Moreover, the substance mentioned, while being known inthe prior art, may not be known as yet as increasing or reversing theactivity of the desaturases. The reaction mixture can be a cell-freeextract or encompass a cell or cell culture. Suitable methods are knownto the skilled worker and are described in general terms for example inAlberts, Molecular Biology the cell, 3rd Edition (1994), for exampleChapter 17. The substances mentioned can be added for example to thereaction mixture or the culture medium or else injected into the cellsor sprayed onto a plant.

When a sample comprising an active substance by the method according tothe invention has been identified, it is either possible directly toisolate the substance from the original sample or else the sample can bedivided into various groups, for example when they consist of amultiplicity of various components, in order to reduce the number of thevarious substances per sample and then to repeat the method according tothe invention with such a “subset” of the original sample. Depending onthe complexity of the sample, the above-described steps can be repeatedrepeatedly, preferably until the sample identified in accordance withthe method according to the invention only still contains a small numberof substances, or only one substance. Preferably, the substanceidentified in accordance with the method according to the invention, orderivatives of the substance, are formulated further so that it issuitable for use in plant breeding or in plant cell or tissue culture.

The substances which have been assayed and identified in accordance withthe method according to the invention can be: expression libraries, forexample cDNA expression libraries, peptides, proteins, nucleic acids,antibodies, small organic substances, hormones, PNAs or the like(Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995),237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein).These substances can also be functional derivatives or analogs of theknown inhibors or activators. Methods of preparing chemical derivativesor analogs are known to the skilled worker. The derivatives and analogsmentioned can be assayed in accordance with prior-art methods. Moreover,computer-aided design or peptidomimetics can be used for producingsuitable derivatives and analogs. The cell or the tissue which can beused for the method according to the invention is preferably a host cellaccording to the invention, a plant cell according to the invention or aplant tissue as described in the abovementioned embodiments.

Accordingly, the present invention also relates to a substance which hasbeen identified in accordance with the above methods according to theinvention. The substance is, for example, a homolog of the desaturasesaccording to the invention. Homologs of the desaturases can be generatedby mutagenesis, for example by point mutation or deletion of thedesaturases. The term “homolog” as used in the present context denotes avariant form of the desaturases which acts as agonist or antagonist forthe activity of the desaturases. An agonist can have essentially thesame or part of the biological activity of the desaturases. Anantagonist of the desaturases can inhibit one or more activities of thenaturally occurring forms of the desaturases, for example can undergocompetitive banding to a downstream or upstream member of the fatty acidsynthesis metabolic pathways, which include the desaturases, or can bindto desaturases and thus reduce or inhibit the activity.

Moreover, the present invention also relates to an antibody or afragment thereof as are described herein, which antibody or fragmentinhibits the activity of the desaturases according to the invention.

In one aspect, the present invention relates to an antibody whichspecifically recognizes, or binds to, the above-described agonist orantagonist according to the invention.

A further aspect relates to a composition comprising the antibody, thestop identified by the method according to the invention or theantisense molecule.

In a further embodiment, the present invention relates to a kitcomprising the nucleic acid according to the invention, the geneconstruct according to the invention, the amino acid sequence accordingto the invention, the antisense nucleic acid molecule according to theinvention, the antibody and/or composition according to the invention,an antagonist or agonist prepared by the method according to theinvention, and/or oils, lipids and/or fatty acids according to theinvention or a fraction thereof. Equally, the kit can comprise the hostcells, organisms, plants according to the invention or parts thereof,harvestable parts of the plants according to the invention orpropagation material or else the antagonist or agonist according to theinvention. The components of the kit of the present invention can bepackaged in suitable containers, for example with or in buffers or othersolutions. One or more of the abovementioned components may be packagedin one and the same container. In addition, or as an alternative, one ormore of the abovementioned components can be adsorbed onto a solidsurface, for example nitrocellulose filters, glass sheets, chips, nylonmembranes or microtiter plates. The kit can be used for any of themethods and embodiments described herein, for example for the productionof host cells, transgenic plants, for the detection of homologoussequences, for the identification of antagonists or agonists and thelike. Furthermore, the kit can comprise instructions for the use of thekit for one of the abovementioned applications.

The present invention is illustrated in greater detail by the exampleswhich follow, and which must not be construed as limiting. The contentof any references, patent applications, patents and published patentapplications cited in the present patent application is herewithincorporated by reference.

EXAMPLES SECTION Example 1 General Methods a) General Cloning Methods:

Cloning methods such as, for example, restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli and yeast cells, the culture ofbacteria and the sequence analysis of recombinant DNA were carried outas described in Sambrook et al. (1989) (Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994)“Methods in Yeast Genetics” (Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3). The transformation and culture of algae such asChlorella or Phaeodactylum are carried out as described by El-Sheekh(1999), Biologia Plantarum 42:209-216; Apt et al. (1996) Molecular andGeneral Genetics 252 (5):872-9.

b) Chemicals

Unless otherwise specified in the text, the chemicals used were obtainedin analytical quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth(Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions weresupplied using pure pyrogen-free water, referred to in the followingtext as H₂O, on a Milli-Q water system water purification unit(Millipore, Eschbom). Restriction endonucleases, DNA-modifying enzymesand molecular biology kits were obtained from AGS (Heidelberg), Amersham(Braunschweig), Biometra (Gottingen), Boehringer (Mannheim), Genomed(Bad Oeynhausen), New England Biolabs (Schwalbach/Taunus), Novagen(Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg),Qiagen (Hilden) and Stratagene (Amsterdam, the Netherlands). Unlessotherwise specified, they were used following the manufacturer'sinstructions.

c) Cell Material

The isolated nucleic acid sequences according to the invention arepresent in the genome of a Phaeodactylum tricornutum UTEX646 strain,which is available from the algae collection of the University of Texas,Austin.

Phaeodactylum tricornutum was cultured at 25° C. at a light/dark photoperiod of 14:10 hours at 22° C. and 35 microEinstein (corresponds tomicromol of photons per square meter and second) in glass tubes intowhich air was passed in from the bottom.

The culture medium used for Phaeodactylum tricornutum was the f/2culture medium supplemented with 10% organic medium of Guillard, R. R.L. (1975; Culture of phytoplankton for feeding marine invertebrates. In:Smith, W. L. and Chanley, M. H. (Eds.) Culture of marine Invertebrateanimals, NY Plenum Press, pp. 29-60.):

It comprises

-   995.5 ml of (artificial) sea water-   1 ml of NaNO₃ (75 g/l), 1 ml of NaH₂PO₄ (5 g/l), 1 ml of trace    element solution, 1 ml of Tris/Cl pH 8.0, 0.5 ml of f/2 vitamin    solution-   Trace element solution: Na₂EDTA (4.36 g/l), FeCl₃ (3.15 g/l),-   Primary trace elements: CuSO₄ (10 g/l), ZnSO₄ (22 g/l), CoCl₂ (10    g/l), MnCl₂ (18 g/l), NaMoO₄ (6.3 g/l)-   f/2 vitamin solution: biotin: 10 mg/l, thiamine 200 mg/l, vitamin B    12 0.1 mg/l-   org medium: sodium acetate (1 g/l), glucose (6 g/l), sodium    succinate (3 g/l), Bacto-tryptone (4 g/l), yeast extract (2 g/l)

Example 2 Isolation of Total DNA from Phaeodactylum tricornutum UTEX646for Hybridization Experiments

The details of the isolation of total DNA refer to the work-up of plantmaterial with a fresh weight of one gram.

CTAB buffer: 2% (w/v) N-acetyl-N,N,N-trimethylammonium bromide (CTAB);100 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA.

N-Laurylsarcosine buffer: 10% (w/v) of N-laurylsarcosine; 100 mMTris-HCl, pH 8.0; 20 mM EDTA.

Phaeodactylum tricornutum cell material was triturated in a mortar underliquid nitrogen to give a fine powder which was transferred into 2 mlEppendorf vessels. The frozen plant material was then covered with alayer of 1 ml of break buffer (1 ml of CTAB buffer, 100 ml ofN-laurylsarcosine buffer, 20 ml of β-mercaptoethanol and 10 ml ofproteinase K solution, 10 mg/ml) and incubated at 60° C. for one hourwith continuous shaking. The homogenate obtained was distributed intotwo Eppendorf vessels (2 ml) and extracted twice by shaking with anequal volume of chloroform/isoamyl alcohol (24:1). For phase separation,centrifugation was carried out at 8 000×g and RT (=room temperature=˜23°C.) for 15 minutes in each case. The DNA was then precipitated for 30minutes at −70° C. using ice-cold isopropanol. The precipitated DNA wassedimented for 30 minutes at 10 000 g at 4° C. and resuspended in 180 mlof TE buffer (Sambrook et al., 1989, Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6). For further purification, the DNA wastreated with NaCl (final concentration 1.2 M) and reprecipitated for 30minutes at −70° C. using twice the volume of absolute ethanol. After awash step with 70% strength ethanol, the DNA was dried and subsequentlytaken up in 50 ml of H₂O+RNase (final concentration 50 mg/ml). The DNAwas dissolved overnight at 4° C., and the RNase cleavage wassubsequently carried out for 1 hour at 37° C. The DNA was stored at 4°C.

Example 3 Isolation of Total RNA and Poly(A)⁺ RNA from Plants andPhaeodactylum tricomutum

Total RNA was isolated from plants such as linseed and oilseed rape andthe like by a method described by Logemann et al. (1987, Anal. Biochem.163, 21). The total RNA from moss can be obtained from protonema tissueusing the GTC method (Reski et al., 1994, Mol. Gen. Genet.,244:352-359).

RNA Isolation of Phaeodactylum tricornutum:

Frozen samples of algae (−70° C.) were triturated in an ice-cold mortarunder liquid nitrogen to give a fine powder. 2 volumes of homogenizationmedium (12.024 g of sorbitol, 40.0 ml of 1M Tris-HCl, pH 9 (0.2 M); 12.0ml of 5 M NaCl (0.3 M), 8.0 ml of 250 mM EDTA, 761.0 mg of EGTA, 40.0 mlof 10% SDS were made up to 200 ml with H₂O and the pH was brought to8.5) and 4 volumes of phenol with 0.2% mercaptoethanol were added to thefrozen cell powder at 40 to 50° C. while mixing thoroughly. Then, 2volumes of chloroform were added and the mixture was stirred vigorouslyfor 15 minutes. The mixture was centrifuged for 10 minutes at 10 000 gand the aqueous phase was extracted with phenol/chloroform (2 volumes)and subsequently extracted with chloroform.

The resulting volume of the aqueous phase was treated with 1/20 volumeof 4 M sodium acetate (pH 6) and 1 volume of isopropanol (ice-cold), andthe nucleic acids were precipitated overnight at −20° C. The mixture wasthen centrifuged for 30 minutes at 10 000 g and the supernatant pipettedoff. This was followed by a wash step with 70% strength EtOH and anothercentrifugation. The sediment was taken up in Tris-borate buffer (80 mMTris-borate buffer, 10 mM EDTA, pH 7.0). The supernatant was thentreated with ⅓ volume of 8 M LiCl, mixed and incubated for 30 minutes at4° C. After recentrifugation, the sediment was washed with 70% strengthethanol, centrifuged and the sediment was dissolved in RNase-free water.

Poly(A)⁺ RNA was isolated using Dyna Beads® (Dynal, Oslo, Norway)following the instructions in the manufacturer's protocol.

After the RNA or poly(A)⁺ RNA concentration had been determined, the RNAwas precipitated by adding 1/10 volume of 3 M sodium acetate, pH 4.6,and 2 volumes of ethanol and stored at −70° C.

For the analysis, 20 μg portions of RNA were separated in aformaldehyde-containing 1.5% strength agarose gel and transferred tonylon membranes (Hybond, Amersham). Specific transcripts were detectedas described by Amasino ((1986) Anal. Biochem. 152, 304)).

Example 4 Construction of the cDNA Library

To construct the cDNA library from Phaeodactylum tricornutum, thefirst-strand synthesis was carried out using murine leukaemia virusreverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T) primers,while the second-strand synthesis was carried out by incubation with DNApolymerase I, Klenow enzyme and RNase H cleavage at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was quenched byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double-stranded DNA molecules were made blunt-ended with T4 DNApolymerase (Roche, Mannheim) at 37° C. (30 minutes). The nucleotideswere removed by extraction with phenol/chloroform and Sephadex G50 spincolumns. EcoRI/XhoI adapters (Pharmacia, Freiburg, Germany) were ligatedto the cDNA ends by means of T4 DNA ligase (Roche, 12° C., overnight),recut with XhoI and phosphorylated by incubation with polynucleotidekinase (Roche, 37° C., 30 min) This mixture was subjected to separationon a low-melting agarose gel. DNA molecules with over 300 base pairswere eluted from the gel, extracted with phenol, concentrated on ElutipD columns (Schleicher and Schüll, Dassel, Germany) and ligated to vectorarms and packaged into lambda-ZAP-Express phages using the Gigapack Goldkit (Stratagene, Amsterdam, the Netherlands), using the manufacturer'smaterial and following their instructions.

Example 5 DNA Sequencing and Computer Analysis

cDNA libraries as described in Example 4 were used for DNA sequencing bystandard methods, in particular the chain termination method using theABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit(Perkin-Elmer, Weiterstadt, Germany). Sequencing of random clones whichhad been singled out was carried out following preparative plasmidpreparation from cDNA libraries via in-vivo mass excision andretransformation of DH10B on agar plates (details on materials andprotocol: Stratagene, Amsterdam, the Netherlands). Plasmid DNA wasprepared from E. coli cultures grown overnight in Luria brothsupplemented with ampicillin (see Sambrook et al. (1989) (Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6)) using a Qiagen DNApreparation robot (Qiagen, Hilden) following the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

(SEQ ID NO: 32) 5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO: 33)5′-CTAAAGGGAACAAAAGCTG-3′ (SEQ ID NO: 34) 5′-TGTAAAACGACGGCCAGT-3′

The sequences were processed and annotated using the EST-MAX standardsoftware package, which is commercially available from Bio-Max (Munich,Germany). Exploiting comparative algorithms, and using the searchsequence shown in SEQ ID NO: 8, homologous genes were searched for usingthe BLAST program (Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402). Two sequences from Phaeodactylum tricornutum withhomologies with the Physcomitrella patens search sequence werecharacterized in greater detail.

Example 5a Isolation of Phaeodactylum tricornutum Desaturases viaPolymerase Chain Reaction with the Aid of Degenerate Oligonucleotides

Published desaturases allow motifs to be identified which are typical ofΔ5- and Δ6-desaturases. Oligonucleotide sequences with possiblevariations are shown in the following text. Underneath theoligonucleotide sequence, the amino acid from which the base combinationcan be derived is shown in the one-letter code. For example, A/G meansthat either an A or a G is randomly incorporated at this position in theoligonucleotide when the unit is synthesized, since the base tripletderived from the corresponding amino acid can either be AAA or AAG. TheDNA sequence may also contain an inosine (i) if the determination of abase at this position permits three or four different bases owing to thegenetic code. The following sequences and primers can be used:

5 ′-forward primer: F1a: (SEQ ID NO: 35)TGG TGG AA A/G TGG  AAi    CA T/C  AA F1b: (SEQ ID NO: 36)TGG TGG AA A/G TGG  ACi    CA T/C  AA F1a: (SEQ ID NO: 37) W   W   K      W   N/T      H     K/N F1b: (SEQ ID NO: 38) W   W   K      W    K       H     K/N F2a: (SEQ ID NO: 39) Gi TGG AA A/G GAi  A/C Ai CA T/C  AA F2b: (SEQ ID NO: 40) Gi TGG AA A/G TTG  A/C Ai CA T/C  AA F2a: (SEQ ID NO: 41)G/W  W   K     E/D  K/Q/N   H      K/N F2b: (SEQ ID NO: 42)G/W  W   K      W   K/Q/N   H      K/N F3a: (SEQ ID NO: 43)T A/T i    TTG  AAi  A/C A A/G C/A G/A i CA F3b: (SEQ ID NO: 44)T A/T i    TTG  AAi  A/C A A/G CAi       CA F3a: (SEQ ID NO: 45)W          W    K/N  H/N       R/Q       H F3b: (SEQ ID NO: 46)Y          W    K/N  H/N       R/Q       H F4a: (SEQ ID NO: 47)      GTi TGG A A/T G/A GA A/G    CA A/G CA F4b: (SEQ ID NO: 48)      GTi TGG A A/T G/A A/T A T/C CA A/G CA F4a: (SEQ ID NO: 49)     V    W     K/M      E          Q    H F4b: (SEQ ID NO: 50)     V    W     K/M      N/Y        Q    H F5a1: (SEQ ID NO: 51)     CA T/C  TA T/C TGG AA A/G AA T/C CA G C F5a1: (SEQ ID NO: 52)     CA T/C  TA T/C TGG AA A/G AA T/C CA A C F5al: (SEQ ID NO: 53)     H       Y      W   K      N      Q   H/Q F6a: (SEQ ID NO: 54)TTG  TTG  AAi  A/C  A A/G  AA i       CA T/C AA F6a: (SEQ ID NO: 55)W    W    K/N  H/N         K/N        H     K/N 3′-reverse primer R1b:(SEQ ID NO: 56)      GG A/G AA  iAG   G/A  TG G/A TG   T/C TC R1b:(SEQ ID NO: 57)      GG A/G AA  iAA   G/A  TG G/A TG   T/C TC R1a:(SEQ ID NO: 58)      P      F    L         H      H        E R1b:(SEQ ID NO: 59)      P      F    F         H      H        E R2a1:(SEQ ID NO: 60) AA    iAG A/G TG A/G  TG    iA C/T  iA/G  T/C TG R2a2:(SEQ ID NO: 61) AA T/C AA A/G TG A/G  TG    iA C/T  iA/G  T/C TG R2a1:(SEQ ID NO: 62) F      L      H       H      V/I      V/A     Q R3a1:(SEQ ID NO: 63) AT  iTG    iGG  A/G AA  iAA    A/G TG A/G  TG R3a2:(SEQ ID NO: 64) AT  A/G TT iGG  A/G AA  iAA    A/G TG A/G  TG R3a3:(SEQ ID NO: 65) AT  iTG    iGG  A/G AA  iAG    A/G TG A/G  TG R3a4:(SEQ ID NO: 66) AT  A/G TT iGG  A/G AA  iAG    A/G TG A/G  TG R3a1:(SEQ ID NO: 67) I/M H/Q     P    F       F         H       H R3a2:(SEQ ID NO: 68) I/M  N      P    F       L         H       H R4al:(SEQ ID NO: 69)       CT   iGG  A/G AA   iA A/G A/G TG   A/G  TG R4a2:(SEQ ID NO: 70)       GA   iGG  A/G AA   iA A/G A/G TG   A/G  TG R4a3:(SEQ ID NO: 71)       GT   iGG  A/G AA   iA A/G A/G TG   A/G  TG R4a1:(SEQ ID NO: 72) =    T/R/S  P    F        F/L        H        H_ R5a1:(SEQ ID NO: 73) AA iAA     A/G TG A/G TG   T/C TC     T/A/G  AT  T/C TGR5a2: (SEQ ID NO: 74)AA iAG     A/G TG A/G TG   T/C TC     T/A/G  AT  T/C TG R5a1:(SEQ ID NO: 75)  F  F           H      H     E         I           QR5a2: (SEQ ID NO: 76) F  L           H      H     E         I           Q R6a1:(SEQ ID NO: 77) T          iGG iA A/G iAA  A/G TG  A/G TG    iAC R6a1:(SEQ ID NO: 78) T          iGG iA A/G iAG  A/G TG  A/G TG    iAC R6a1:(SEQ ID NO: 79) T/N          P   L    F/L     H       H       V

Owing to various possibilities of variations, a large number of derivedoligonucleotides are possible, but surprisingly it has been found thatoligonucleotides shown can be particularly suitable for isolatingdesaturases.

The primers can be employed for polymerase chain reactions in allcombinations. Individual combinations allowed desaturase fragments to beisolated when the following conditions were taken into consideration:for PCR reactions, in each case 10 nMol of primer and 10 ng of a plasmidlibrary obtained by in-vivo excision were employed. It was possible toisolate the plasmid library from the phage library following theprotocols of the manufacturer (Stratagene). The PCR reaction was carriedout in a thermocycler (Biometra) using Pfu DNA polymerase (Stratagene)and the following temperature program: 3 minutes at 96° C. followed by35 cycles with 30 seconds at 96° C., 30 seconds at 55° C. and 1 minuteat 72° C. After the first step at 55° C., the annealing temperature waslowered stepwise by in each case 3° C., and, after the fifth cycle, anannealing temperature of 40° C. was retained. Finally, a 10-minute-cycleat 72° C. was carried out, and the reaction was stopped by cooling to 4°C.

The primer combinations F6a and R4a2 are shown underlined in the text,and it was possible to exploit them successfully for isolating adesaturase fragment. It was possible to verify the resulting fragment bysequencing; it showed homologies with Streptomyces coelicolor desaturasewith the Genbank Accession No. T36617. The homology was obtained withthe aid of the BLASTP program. The alignment is shown in FIG. 4.Identities of 34% and a homology of 43% with sequence 136617 wererevealed. The DNA fragment was employed in accordance with the inventionas shown in Example 7 in a hybridization experiment for isolating afull-length gene under standard conditions.

The coding region of a DNA sequence isolated in this way was obtained bytranslating the genetic code into a polypeptide sequence. SEQ ID NO: 3shows a sequence 1434 base pairs in length which was isolated by themethod described. The sequence has a start codon in positions 1 to 3 anda stop codon in positions 1432-1434 and it was possible to translate itinto a polypeptide 477 amino acids in length. In the alignment with thegene sequence described in WO 98/46763, it was found that anonidentical, but homologous, Phaeodactylum tricornutum fragmentencoding 87 amino acids had previously been described. However, WO98/46763 discloses neither a complete, functionally active desaturasenor position or substrate specificity. This is also made clear by thefact that homologies with both the Δ5- and Δ6-desaturase fromMortierella alpina are reported without indicating a specific function.The sequence according to the invention, in contrast, encodes afunctionally active Δ6-acyl lipid desaturase.

Example 6 Identification of DNA Sequences Encoding Phaeodactylumtricornutum Desaturases

The full-length sequence of the Δ6-acyl lipid desaturase Pp_des6AJ222980 (NCBI Genbank Accession No.) from the moss Physcomitrellapatens (see also Table 1) and the Δ12-acyl lipid desaturase sequence(Table 1, see Ma_des12) from Mortierella alpina AF110509 (AF110509 NCBIGenbank Accession No.) were employed for sequence alignment with the aidof the TBLASTN search algorithm.

The EST sequences PT0010070010R, PT001072031R and PT001078032R werefirst considered as target gene among further candidate genes owing toweak homologies with the search sequences from Physcomitrella andMortierella. FIGS. 1, 2 and 2 a show the result of the two EST sequencesfound. The sequences found are part of the nucleic acids according tothe invention of SEQ ID NO: 1 (gene name: Pt_des5, inventors' owndatabase No. PT001078032R), SEQ ID NO: 5. (gene name: Pt_des12,inventors' own database No. PT0010070010R) and SEQ ID NO: 11 (gene name:Pt_des12.2, inventor's own database No. PT001072031R). Letters indicateidentical amino acids, while the plus symbol indicates a chemicallysimilar amino acid. The identities and homologies of all sequences foundin accordance with the invention can be seen from the summary in Table2.

Desaturases can have cytochrome b5 domains which also occur in othergenes which do not code for desaturases. Thus, cytochrome b5 domainsshow high homologies, even though the gene functions are different.Within weakly conserved regions, desaturases can only be identified asputative candidate genes and must be tested for the enzyme activity andposition specificity of the enzymatic function. For example, varioushydroxylases, acetylenases and epoxygenases, like desaturases, also showhistidine box motifs, so that a specific function must be provenexperimentally and only the additional verification of the double bondmakes possible a guaranteed enzyme activity and position specificity ofa desaturase. Surprisingly, it has been found that Δ6- andΔ5-desaturases according to the invention have particularly suitablesubstrate specificities and are particularly suitable for beingexploited, in combination with a Physcomitrella Δ6-elongase, forproducing polyunsaturated fatty acids such as arachidonic acid,eicosapentaenoic acid and docosahexanoic acid.

Sequencing of the full cDNA fragment from clone PT001078032R revealed asequence 1652 base pairs in length. The sequence encodes a polypeptideof 469 amino acids shown in SEQ ID NO: 2. It was obtained by translatingthe genetic code of SEQ ID NO: 1 with a start codon in base pairposition 115-117 and with a stop codon in base pair position 1522-1524.The clone comprises a complete desaturase polypeptide, as can be seenfrom the sequence alignment in FIG. 3. Lines denote identical aminoacids, while colons and dots represent chemically exchangeable, i.e.chemically equivalent, amino acids. The alignment was carried out usingHenikoff & Henikoff s BLOSUM62 substitution matrix ((1992) Amino acidsubstitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA89: 10915-10919). Parameters used: Gap Weight: 8; Average Match: 2.912,Length Weight: 2, Average Mismatch: −2.003.

FIG. 6 and FIG. 7 show the alignment of the MA_des12 peptide sequencewith the sequences found.

Sequencing of the complete cDNA fragment from clone PT001007001ORrevealed a sequence 1651 base pairs in length and shown in SEQ ID NO: 5with a start codon in position 67-69 and a stop codon in position1552-1554. The polypeptide sequence according to the invention is shownin SEQ ID NO: 6.

Sequencing the complete cDNA fragment identified from clonePT0010072031R revealed a sequence 1526 base pairs in length and shown inSEQ ID NO: 11 with a start codon in position 92-94 and a stop codon inposition 1400-1402. The polypeptide sequence according to the inventionis shown in SEQ ID NO: 12.

Table 2 shows the identities and homologies of desaturases according tothe invention with each other and with the Physcomitrella patens andMortierella alpina desaturase. The data were obtained with the aid ofthe Bestfit program under given parameters as defined hereinbelow as asubprogram of the following software: Wisconsin Package Version 10.0(Genetics Computer Group (GCG), Madison, Wis., USA). Henikoff, S. andHenikoff, J. G. (1992). Amino acid substitution matrices from proteinblocks. Proc. Natl. Acad. Sci. USA 89: 10915-10919.

Furthermore, FIG. 5 shows the alignment of the Physcomitrella patensΔ6-acyl lipid desaturase with the polypeptide sequence of clone Pt_des6.

TABLE 2 Homology/ Search Search identity sequence sequence in % Pp_des6Ma_des12 Pt_des5 34.92/26.37 n.d. Pt_des6 50.69/41.06 n.d. Pt_des12 n.d.48.58/38.92 Pt_des12.2 n.d. 48.37/41.60 n.d. = not determined

With the aid of the algorithm TBLASTN 2.0.10: Altschul et al. 1997,“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402, sequences with the highestsequence homology or identity were identified via a local databasealignment. The results are shown in Table 2A hereinbelow.

TABLE 2A Homologs with the highest sequence homologies or identitieswith polypeptide sequences according to the invention of SEQ ID NO. 2,4, 6 or 12 Search Search Search Search Homology/ sequence sequencesequence sequence identity (%) PT001070010R PT001072031R PT001078032RPT_des6 L26296: Fad2 50%/37% n.d. n.d. n.d. A. thaliana U86072 n.d.51/40 n.d. n.d. Petroselinum crispum Fad2 AL358652 n.d. n.d. 45/30 n.d.L. major putative desaturase AB020032 n.d. n.d. n.d. 53/38 M. alpina Δ6desaturase

Example 7 Identification of Genes by Means of Hybridization

Gene sequences can be used for identifying homologous or heterologousgenes from cDNA or genomic libraries.

Homologous genes (i.e. full-length cDNA clones which are homologous, orhomologs) can be isolated via nucleic acid hybridization using, forexample, cDNA libraries: the method can be used in particular forisolating functionally active full-length genes of those shown in SEQ IDNO: 3. Depending on the frequency of the gene of interest, 100 000 up to1 000 000 recombinant bacteriophages are plated out and transferred to anylon membrane. After denaturation with alkali, the DNA was immobilizedon the membrane, for example by UV crosslinking. Hybridization isperformed under high-stringency conditions. The hybridization and thewash steps are carried out in aqueous solution at an ionic strength of 1M NaCl and a temperature of 68° C. Hybridization probes were generatedfor example by labeling with radioactive (³²P-) nick transcription (HighPrime, Roche, Mannheim, Germany). The signals are detected byautoradiography.

Partially homologous or heterologous genes which are related but notidentical can be identified analogously to the process described aboveusing low-stringency hybridization and wash conditions. For the aqueoushybridization, the ionic strength was usually kept at 1 M NaCl, and thetemperature was lowered gradually from 68 to 42° C.

The isolation of gene sequences which only exhibit homologies with anindividual domain of, for example, 10 to 20 amino acids can be carriedout using synthetic, radiolabeled oligonucleotide probes. Radiolabeledoligonucleotides are generated by phosphorylating the 5′ end of twocomplementary oligonucleotides with T4 polynucleotide kinase. Thecomplementary oligonucleotides are hybridized and ligated to each otherto give rise to concatemers. The double-stranded concatemers areradiolabeled for example by nick transcription. Hybridization is usuallycarried out under low-stringency conditions using high oligonucleotideconcentrations.

Oligonucleotide Hybridization Solution:

-   6×SSC-   0.01 M sodium phosphate-   1 mM EDTA (pH 8)-   0.5% SDS-   100 μg/ml denatured salmon sperm DNA-   0.1% dry low-fat milk

During the hybridization, the temperature is lowered stepwise to 5 to10° C. below the calculated oligonucleotide Tm or down to roomtemperature (unless otherwise specified, RT=˜23° C. in all experiments),followed by wash steps and autoradiography. Washing is carried out atextremely low stringency, for example three wash steps using 4×SSC.Further details are as described by Sambrook, J., et al. (1989),“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, or Ausubel, F. M., et al. (1994) “Current Protocols in MolecularBiology”, John Wiley & Sons.

Example 8 Identification of Target Genes by Screening ExpressionLibraries with Antibodies

To generate recombinant protein, for example E. coli, cDNA sequenceswere used (for example Qiagen QIAexpress pQE system). The recombinantproteins were then affinity-purified, usually via Ni-NTA affinitychromatography (Qiagen). The recombinant proteins were then used forraising specific antibodies, for example using standard techniques forimmunizing rabbits. The antibodies were then affinity-purified using anNi-NTA column which is desaturated with recombinant antigen, asdescribed by Gu et al., (1994) BioTechniques 17:257-262. The antibodycan then be used for screening expression cDNA libraries byimmunological screening (Sambrook, J., et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, orAusubel, F. M., et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 9 Transformation of Agrobacterium

Agrobacterium-mediated plant transformation can be effected for exampleusing the Agrobacterium tumefaciens strain GV3101- (pMP90-) (Koncz andSchell, Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404- (Clontech) orC58C1 pGV2260 (Deblaere et al 1984, Nucl. Acids Res. 13, 4777-4788). Thetransformation can be carried out by standard transformation techniques(Deblaere et al., 1984, IBID.).

Example 10 Plant Transformation

Agrobacterium-mediated plant transformation can be effected usingstandard transformation and regeneration techniques (Gelvin, Stanton B.,Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd Ed.,Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc ZentraleSignatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, JohnE., Methods in Plant Molecular Biology and Biotechnology, Boca Raton:CRC Press, 1993, 360 pp., ISBN 0-8493-5164-2).

For example, oilseed rape can be transformed by means of cotyledon orhypocotyl transformation (Moloney et al., Plant Cell 8 (1989) 238-242;De Block et al., Plant Physiol. 91 (1989) 694-701). The use ofantibiotics for the selection of agrobacteria and plants depends on thebinary vector and the agrobacterial strain used for the transformation.The selection of oilseed rape is normally carried out using kanamycin asselectable plant marker.

Agrobacterium-mediated gene transfer in linseed (Linum usitatissimum)can be carried out for example using a technique described by Mlynarovaet al. (1994) Plant Cell Report 13:282-285.

The transformation of soybean can be carried out for example using atechnique described in EP-A-0 0424 047 (Pioneer Hi-Bred International)or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770(University Toledo).

Plant transformation using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling and Walbot “The maize handbook”(1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 11 Plasmids for Plant Transformation

Binary vectors such as pBinAR (Höfgen and Willmitzer, Plant Science 66(1990) 221-230) or pGPTV (Becker et al 1992, Plant Mol. Biol.20:1195-1197) or derivatives of these can be used for transformingplants. The binary vectors can be constructed by ligating the cDNA insense or antisense orientation into T-DNA. 5′ of the cDNA, a plantpromoter activates cDNA transcription. A polyadenylation sequence islocated 3′ of the cDNA. The binary vectors can contain different markergenes. In particular, the nptII marker gene encoding kanamycinresistance mediated by neomycin phosphotransferase can be exchanged forthe herbicide-resistant form of an acetolactate synthase gene(abbreviation: AHAS or ALS). The ALS gene is described in Ott et al., J.Mol. Biol. 1996, 263:359-360. The v-ATPase-c1 promoter can be clonedinto plasmid pBin19 or pGPTV and exploited for marker gene expression bycloning it before the ALS coding region. The promoter stated correspondsto a 1153 base pair fragment from Beta vulgaris (Plant Mol. Biol., 1999,39:463-475). Both sulfonylureas and imidazolinones such as imazethapyror sulfonylureas can be used as antimetabolites for selection.

Tissue-specific expression can be achieved by using a tissue-specificpromoter. For example, seed-specific expression can be achieved bycloning the DC3 or the LeB4 or the USP promoter or the phaseolinpromoter 5′ of the cDNA. Any other seed-specific promoter element canalso be used, such as, for example, the napin or arcelin promoter(Goossens et al. 1999, Plant Phys. 120(4):1095-1103 and Gerhardt et al.2000, Biochimica et Biophysica Acta 1490(1-2):87-98). The CaMV 35Spromoter or a v-ATPase C1 promoter can be used for constitutiveexpression in imtact plants.

In particular, genes encoding desaturases and elongases can be clonedinto a binary vector one after the other by constructing severalexpression cassettes in order to imitate the metabolic pathway inplants.

Within an expression cassette, the protein to be expressed can betargeted into a cellular compartment using a signal peptide, for examplefor plastids, mitochondria or the endoplasmic reticulum (Kermode, Crit.Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is cloned 5′in-frame with the cDNA in order to achieve subcellular localization ofthe fusion protein.

Examples of multiexpression cassettes are given hereinbelow.

I.) Promoter-Terminator Cassettes

Expression cassettes are composed of at least two functional units, suchas a promoter and a terminator. Further desired gene sequences such astargeting sequences, coding regions of genes or parts thereof etc. canbe inserted between promoter and terminator. In order to constructexpression cassettes, promoters and terminators (USP promoter: Baeumleinet al., Mol. Gen. Genet., 1991, 225 (3):459-67); OCS terminator: Gielenet al. EMBO J. 3 (1984) 835 et seq.) are isolated with the aid ofpolymerase chain reaction and tailor-made as desired with flankingsequences based on synthetic oligonucleotides.

Examples of the oligonucleotides which can be used are the following:

USP1 front: (SEQ ID NO: 80)CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP2 front:(SEQ ID NO: 80) CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCAUSP3 front: (SEQ ID NO: 80)CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP1 back:(SEQ ID NO: 81) AAAACTGCAGGCGGCCGCCCACCGCGGTGGGCTGGCTATGAAGAAATTUSP2 back: (SEQ ID NO: 82) CGCGGATCCGCTGGCTATGAAGAAATT USP3 back:(SEQ ID NO: 83) TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATTOCS1 front: (SEQ ID NO: 84) AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATATOCS2 front: (SEQ ID NO: 85)CGCGGATCCGATATCGGGCCCGCTAGCGTTAACCCTGCTTTAATGAG ATAT OCS3 front:(SEQ ID NO: 86) TCCCCCGGGCCATGGCCTGCTTTAATGAGATAT OCS1 back:(SEQ ID NO: 87) CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAA ATTGAOCS2 back: (SEQ ID NO: 87)CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAA ATTGA OCS3 back:(SEQ ID NO: 88) CCCAAGCTTGGCGCGCCGAGCTCGTCGACGGACAATCAGTAAATTGA

The methods are known to the skilled worker in the art and are generallyknown from the literature.

In a first step, a promoter and a terminator are amplified via PCR.Then, the terminator is cloned into a recipient plasmid and, in a secondstep, the promoter is inserted before the terminator. This gives anexpression cassette on a carrier plasmid. Plasmids pUT1, pUT2 and pUT3are generated on the basis of plasmid pUC19.

The constructs are defined in accordance with the invention in SEQ IDNO: 13, 14 and 15. Based on pUC19, they comprise the USP promoter andthe OCS terminator. Based on these plasmids, construct pUT12 isgenerated by cutting pUT1 with SalI/ScaI and cutting pUT2 withXhoI/ScaI. The fragments in the expression cassette are ligated andtransformed into E. coli XLI blue MRF. After singling outampicillin-resistant colonies, DNA is prepared, and those clones, whichcomprise two expression cassettes are identified by restrictionanalysis. The XhoI/SalI ligation of compatible ends has eliminated thetwo cleavage sites XhoI and SalI between the expression cassettes. Thisgives rise to plasmid pUT12, which is defined in SEQ ID NO: 16. pUT12 issubsequently cut again with SalI/ScaI and pUT3 with XhoI/ScaI. Thefragments comprising the expression cassettes are ligated andtransformed into E. coli XLI blue MRF. After singling outampicillin-resistant columns, DNA is prepared, and those clones whichcomprise three expression cassettes are identified by restrictionanalysis. In this manner, a set of multiexpression cassettes is createdwhich can be exploited for inserting the desired DNA and is described inTable 3 and can additionally incorporate further expression cassettes.

They comprise the following elements:

TABLE 3 pUC19 Cleavage sites for Multiple cloning Cleavage sites behindderivate the USP promoter cleavage sites the OCS terminator pUT1EcoRI/AscI/SacI/XhoI BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/SacI/AscI/HindIII pUT2 EcoRI/AscI/SacI/XhoIBamHI/EcoRV/ApaI/NheI/HpaI SalI/EcoRI/SacI/AscI/HindIII pUT3EcoRI/AscI/SacI/XhoI BglII/NaeI/ClaI/SmaI/NcoI SalI/SacI/AscI/HindIIIpUT12 EcoRI/AscI/SacI/XhoI BstXI/NotI/PstII/XbaI/StuI andSalI/EcoRI/SacI/AscI/HindIII Double expressionBamHI/EcoRV/ApaI/NheI/HpaI cassette pUT123 EcoRI/AscI/SacI/XhoI 1.BstXI/NotI/PstI/XbaI/StuI and SalI/SacI/AscI/HindIII Triple expression2. BamHI/EcoRV/ApaI/NheI/HpaI and cassette 3. BglII/NaeI/ClaI/SmaI/NcoI

Furthermore, further multiexpression cassettes can be generated andemployed for the seed-specific gene expression, as described and asspecified in greater detail in Table 4, with the aid of the

-   i) USP promoter or with the aid of the-   ii) approx. 700 base pair 3′ fragment of the LeB4 promoter or with    the aid of the-   iii) DC3 promoter.

The DC3 promoter is described in Thomas, Plant Cell 1996, 263:359-368and consists merely of the region −117 to +27, which is why it thereforeconstitutes one of the smallest known seed-specific promoters. Theexpression cassettes can comprise several copies of the same promoter orelse be constructed via three different promoters.

TABLE 4 Multiple expression cassettes Plasmid name of the pUC19 Cleavagesites before Multiple cloning Cleavage sites behind derivative therespective promoter Cleavage sites the OCS terminator pUT1EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuISalI/EcoRI/SacI/AscI/HindIII (pUC19 with USP-OCS1) pDCTEcoRI/AscI/SacI/XhoI (2) BamHI/EcoRV/ApaI/NheI/HpaISalI/EcoRI/SacI/AscI/HindIII (pUC19 with DC3-OCS) pLeBTEcoRI/AscI/SacI/XhoI (3) BglII/NaeI/ClaI/SmaI/NcoISalI/SacI/AscI/HindIII (pUC19-with LeB4(700)-OCS) pUD12EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI andSalI/EcoRI/SacI/AscI/HindIII (pUC 19 with (2) BamHI/EcoRV/ApaI/NheI/HpaIUSP-OCS1 and with DC3-OCS) pUDL123 EcoRI/AscI/SacI/XhoI (1)BstXI/NotI/PstI/XbaI/StuI and SalI/SacI/AscI/HindIII Triple expression(2) BamHI/(EcoRV*)/ApaI/NheI/HpaI and cassette (pUC19 (3)BglII/NaeI/ClaI/SmaI/NcoI with USP/DC3 and LeB4-700) *EcoRV cleavagesite in the 700 base-pair fragment of the LeB4 promoter (LeB4-700).

Further promoters for multi-gene constructs can be generatedanalogously, in particular using the

-   a) 2.7 kb fragment of the LeB4 promoter or with the aid of the-   b) phaseolin promoter or with the aid of the-   c) constitutive v-ATPase c1 promoter.

It may be particularly desirable to use further especially suitablepromoters for constructing seed-specific multi-expression cassettes suchas, for example, the napin promoter or the arcelin-5 promoter.

-   ii) Generation of Expression Construct in pUC19 Derivatives or pGPTV    Derivatives Receiving Promoter and Terminator and Comprised in    Combination with Desired Gene Sequences for PUFA Gene Expression in    Plant Expression Cassettes.

Using AscI, multi-expression cassettes can be inserted directly frompUC19 derivatives of Table 3 into the vector pGPTV+AscI (see iii.)) viathe AscI cleavage site and are available for inserting target genes. Thegene constructs in question (pBUT1 is shown in SEQUENCE ID NO: 20, pBUT2is shown in SEQUENCE ID NO: 21, pBUT3 is shown in SEQUENCE ID NO: 22,pBUT 12 is shown in SEQUENCE ID NO: 22 and pBUT123 is shown in SEQUENCEID NO: 24) are available in accordance with the invention in kit form.As an alternative, gene sequences can be inserted into the pUC19-basedexpression cassettes and inserted into pGPTV+AscI in the form of an AscIfragment.

In pUT12, the Δ6-elongase Pp_PSE1 is first inserted into the firstcassette via BstXI and XbaI. Then, the moss Δ6-desaturase (Pp_des6) isinserted into the second cassette via BamHI/NaeI. This gives rise to theconstruct pUT-ED. The AscI fragment from plasmid pUT-ED is inserted intothe AscI-cut vector pGPTV+AscI, and the orientation of the insertedfragment is determined by restriction or sequencing. This gives rise toplasmid pB-DHGLA, whose complete sequence is shown in SEQUENCE ID NO.25. The coding region of the Physcomitrella Δ6-elongase is shown inSEQUENCE ID NO. 26, that of the Physcomitrella Δ6-desaturase in SEQUENCEID NO: 27.

In pUT123, the Δ6-elongase Pp_PSE1 is first inserted into the firstcassette via BstXI and XbaI. Then, the moss Δ6-desaturase (Pp_des6) isinverted into the second cassette via BamHI/NaeI, and, finally, thePhaeodactylum Δ5-desaturase (Pt des5) is inserted into the thirdcassette via BglII. The triple construct is given the name pARA1. Takinginto consideration sequence-specific restriction cleavage sites, furtherexpression cassettes termed pARA2, pARA3 and pARA4 can be generated, asshown in Table 5.

The AscI fragment from plasmid pARA1 is inserted into the AscI-cutvector pGPTV+AscI and the orientation of the inserted fragment isdetermined by means of restriction or sequencing. The complete sequenceof the resulting plasmid pBARA1 is shown in SEQUENCE ID NO. 28. Thecoding region of the Physcomitrella Δ6-elongase is shown in SEQUENCE IDNO. 29, that of the Physcomitrella Δ6-desaturase in SEQUENCE ID NO: 30and that of the Phaeodactylum tricornutum Δ5-desaturase in SEQUENCE IDNO: 31.

TABLE 5 Combinations of desaturases and elongases Gene PlasmidΔ6-desaturase Δ5-desaturase Δ6-elongase 1 PUT-ED Pp_des6 — Pp_PSE1 2pARA1 Pt_des6 Pt_des5 Pp_PSE1 3 pARA2 Pt_des6 Ce_des5 Pp_PSEl 4 pARA3Pt_des6 Ce_des5 Pp_PSE1 5 pARA4 Ce_des6 Ce_des5 Ce_PSE1 6 PBDHGLAPt_des6 — Pp_PSE1 7 PBARAI Pt_des6 Pt_des5 Pp_PSE1

Plasmids 1 to 5 are pUC derivatives, plasmids 6 to 7 are binary planttransformation vectors

-   Pp=Physcomitrella patens, Pt=Phaeodactylum tricornutum-   Pp_PSE1 corresponds to the sequence of SEQ ID NO: 9.-   PSE=PUFA-specific Δ6-elongase-   Ce_des5=Caenorhabditis elegans Δ5-desaturase (Genbank Acc. No.    AF078796)-   Ce_des6=Caenorhabditis elegans Δ6-desaturase (Genbank Acc. No.    AF031477, bases 11-1342)-   CePSE1=Caenorhabditis elegans Δ6-elongase (Genbank Acc. No.    AF244356, bases 1-867)

Further desaturases or elongase gene sequences can also be inserted intoexpression cassettes of the above-described type, such as, for example,Genbank Acc. No. AF231981, NM_(—)013402, AF206662, AF268031, AF226273,AF110510 or AF110509.

-   iii) Transfer of Expression Cassettes into Vectors for the    Transformation of Agrobacterium tumefaciens and for the    Transformation of Plants

Chimeric gene constructs based on those described in pUC19 can beinserted into the binary vector pGPTV by means of AscI. For thispurpose, the multiple cloning sequence is extended by an AscI cleavagesite. For this purpose, the polylinker is newly synthesized as twodouble-stranded oligonucleotides, an additional AscI DNA sequence beinginserted. The oligonucleotide is inserted into the vector pGPTV by meansof EcoRI and HindIII. This gives rise to plasmid pGPTV+AscI. The cloningtechniques required are known to the skilled worker and can simply befound as described in Example 1.

Example 12 In-vivo Mutagenesis

The in-vivo mutagenesis of microorganisms can be performed by passagingthe plasmid DNA (or any other vector DNA) via E. coli or othermicroorganisms (for example Bacillus spp. or yeasts such asSaccharomyces cerevisiae) in which the ability of maintaining theintegrity of the genetic information is disrupted. Conventional mutatorstrains have mutations in the genes for the DNA repair system (forexample mutHLS, mutD, mutT and the like; as reference, see Rupp, W. D.(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, pp.2277-2294, ASM: Washington). These strains are known to the skilledworker. The use of these strains is illustrated for example in Greener,A., and Callahan, M. (1994) Strategies 7:32-34. The transfer of mutatedDNA molecules into plants is preferably effected after themicroorganisms have been selected and tested. Transgenic plants aregenerated in accordance with various examples in the examples section ofthe present document.

Example 13 Studying the Expression of a Recombinant Gene Product in aTransformed Organism

The activity of a recombinant gene product in the transformed hostorganism can be measured at the transcription and/or translation level.

A suitable method for determining the amount of transcription of thegene (which indicates the amount of RNA available for translation of thegene product) is to carry out a Northern blot as specified hereinbelow(for reference, see Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: New York, or the abovementioned examples section) inwhich a primer which is designed such that it binds to the gene ofinterest is labeled with a detectable label (usually radioactivity orchemiluminescent) so that, when the total RNA of a culture of theorganism is extracted, separated on a gel, transferred to a stablematrix and incubated with this probe, binding and the extent of bindingof the probe indicate the presence and also the quantity of the mRNA forthis gene. This information indicates the degree of transcription of thetransformed gene. The total cell RNA can be prepared from cells, tissuesor organs by a plurality of methods, all of which are known in the art,such as, for example, the method of Bormann, E. R., et al. (1992) Mol.Microbiol. 6:317-326.

Northern Hybridization

For the RNA hybridization, 20 μg of total RNA or 1 μg of poly(A)⁺ RNAwere separated by gel electrophoresis in 1.25% strength agarose gelusing formaldehyde as described by Amasino (1986, Anal. Biochem. 152,304), transferred to positively charged nylon membranes (Hybond N+,Amersham, Braunschweig) by capillary attraction using 10×SSC,immobilized by means of UV light and prehybridized for 3 hours at 68° C.using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS,100 mg herring sperm DNA). The DNA probe had been labeled with theHighprime DNA labeling kit (Roche, Mannheim, Germany) during theprehybridization stage using alpha-³²P-dCTP (Amersham, Braunschweig,Germany). The hybridization was carried out after adding the labeled DNAprobe in the same buffer at 68° C. overnight. The wash steps werecarried out twice for 15 minutes using 2×SSC and twice for 30 minutesusing 1×SSC, 1% SDS, at 68° C. The sealed filters were exposed at −70°C. for a period of 1 to 14 days.

Standard techniques such as a Western blot (see, for example, Ausubel etal. (1988) Current Protocols in Molecular Biology, Wiley: New York) canbe employed for studing the presence or the relative quantity of proteintranslated from this mRNA. In this method, the total cell proteins areextracted, separated by means of gel electrophoresis, transferred to amatrix such as nitrocellulose, and incubated with a probe such as anantibody which specifically binds to the desired protein. This probe isusually provided with a chemiluminescent or colorimetric label which canbe detected readily. The presence and the quantity of the label observedindictes the presence and quantity of the desired mutated protein whichis present in the cell.

Example 14 Analysis of the Effect of the Recombinant Proteins on theProduction of the Desired Product

The effect of the genetic modification in plants, fungi, algae, ciliatesor on the production of a desired compound (such as a fatty acid) can bedetermined by growing the modified microorganisms or the modified plantunder suitable conditions (such as those described above) and analyzingthe medium and/or the cell components for the increased production ofthe desired product (i.e. of lipids or a fatty acid). These analyticaltechniques are known to the skilled worker and encompass spectroscopy,thin-layer chromatography, various staining methods, enzymatic andmicrobiological methods, and analytical chromatography such ashigh-performance liquid chromatography (see, for example, Ullman,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III: “Product recovery and purification”, pp. 469-714, VCHWeinheim; Belter, P. A., et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F., andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D. (1988)Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, pp. 1-27, VCH Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned methods, plant lipids are extractedfrom plant materials as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) AnalyticBiochemistry 152:141-145. Qualitative and quantitative lipid or fattyacid analysis is described in Christie, William W., Advances in LipidMethodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2);Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for producing the desired compound, such as intermediates andbyproducts, in order to determine the overall production efficiency ofthe compound. The analytical methods encompass measurements of thenutrient quantities in the medium (for example sugars, hydrocarbons,nitrogen sources, phosphate and other ions), measurements of biomassconcentration and growth, analysis of the production of customarymetabolites of biosynthetic pathways, and measurements of gases whichare generated during fermentation. Standard methods for thesemeasurements are described in Applied Microbial Physiology; A PracticalApproach, P. M. Rhodes and P. F. Stanbury, Ed., IRL Press, S. 103-129;131-163 and 165-192 (ISBN: 0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas-liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

The unambiguous detection of the presence of fatty acid products can beobtained by analyzing recombinant organisms by analytical standardmethods: GC, GC-MS or TLC, as they are described on several occasions byChristie and the references therein (1997, in: Advances on LipidMethodology, Fourth Edition: Christie, Oily Press, Dundee, 119-169;1998, gas chromatography/mass spectrometry methods, Lipide 33:343-353).

The material to be analyzed can be disrupted by ultrasonication,grinding in a glass mill, liquid nitrogen and grinding or by otherapplicable methods. After disruption, the material must be centrifuged.The sediment is resuspended in distilled water, heated for 10 minutes at100° C., ice-cooled and recentrifuged, followed by extraction in 0.5 Msulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90° C.,which leads to hydrolyzed oil and lipid compounds which givetransmethylated lipids. These fatty acid methyl esters are extracted inpetroleum ether and finally subjected to GC analysis using a capillarycolumn (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm) at atemperature gradient between 170° C. and 240° C. for 20 minutes and 5minutes at 240° C. The identity of the resulting fatty acid methylesters must be defined using standards which are commercially available(i.e. Sigma).

In the case of fatty acids for which no standards are available, theidentity must be demonstrated via derivatization followed by GC/MSanalysis. For example, the localization of fatty acids with triple bondsmust be demonstrated via GC/MS following derivatization with4,4-dimethoxyoxazolin derivatives (Christie, 1998, see above).

Expression Constructs in Heterologous Microbial System Strains, WashConditions and Plasmids

The Escherichia coli strain XL1 Blue MRF' kan (Stratagene) was used forsubcloning novel Physcomitrella patens desaturase pPDesaturase1. Forfunctionally expressing this gene, we used the Saccharomyces cerevisiaestrain INVSc 1 (Invitrogen Co.). E. coli was grown in Luria-Bertinibroth (L B, Duchefa, Haarlem, the Netherlands) at 37° C. If necessary,ampicillin (100 mg/liter) was added, and 1.5% of agar (w/v) was addedfor solid LB media. S. cerevisiae was grown at 30° C. either in YPGmedium or in complete minimal dropout uracil medium (CMdum; see in:Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., Struhl, K., Albright, L. B., Coen, D. M., and Varki,A. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork) together with 2% (w/v) of either raffinose or glucose. For solidmedia, 2% (w/v) of Bacto™-agar (Difco) were added. The plasmids used forcloning an expression are pUC 18 (Pharmacia) and pYES2 (Invitrogen Co.).

Example 16 Cloning and Expression of PUFA-Specific Phaeodactylumtricornutum Desaturases

For the expression in yeast, the Phaeodactylum tricornutum cDNA clonesfrom SEQ ID NO: 1, 3, 5 or 11 or the sequences from SEQ ID NO: 7 or 9 orother desired sequences were first modified in such a way that only thecoding regions are amplified by means of polymerase chain reaction withthe aid of two oligonucleotides. Care was taken that a consensussequence for the start codon was retained for efficient translation. Tothis end, either the base sequence ATA or AAA was selected and insertedinto the sequence before the ATG (Kozak, M. (1986) Point mutationsdefine a sequence flanking the AUG initiator codon that modulatestranslation by eukaryotic ribosomes, Cell 44, 283-292). A restrictioncleavage site was additionally introduced before this consensus triplet,which restriction cleavage site must be compatible with the cleavagesite of the target vector into which the fragment is to be cloned andwith the aid of which gene expression is to take place in microorganismsor plants.

The PCR reaction was carried out with plasmid DNA as template in athermocycler (Biometra) using Pfu DNA (Stratagene) polymerase and thefollowing temperature program: 3 minutes at 96° C., followed by 30cycles with 30 seconds at 96° C., 30 seconds at 55° C. and 2 minutes at72° C., 1 cycle with 10 minutes at 72° C. and stop at 4° C. Theannealing temperature was varied depending on the oligonucleotideschosen. A synthesis time of approximately one minute can be assumed perkilobase pairs of DNA. Further parameters which have an effect on thePCR, such as, for example, Mg ions, salt, DNA polymerase and the likeare known to the skilled worker and can be varied as required.

The correct size of the amplified DNA fragment was controlled by meansof agarose TBE gel electrophoresis. The amplified DNA was extracted fromthe gel using the QIAquick gel extraction kit (QIAGEN) and ligated intothe SmaI restriction site of the dephosphorylated vector pUC18 using theSure Clone Ligations Kit (Pharmacia), giving rise to the pUCderivatives. Following the transformation of E. coli XL1 Blue MRF' kan,a DNA mini preparation (Riggs, M. G., & McLachlan, A. (1986) Asimplified screening procedure for large numbers of plasmidmini-preparation. BioTechniques 4, 310-313) was carried out onampicillin-resistant transformants, and positive clones were identifiedby means of BamHI restriction analysis. The sequence of the cloned PCRproduct was confirmed by resequencing using the ABI PRISM Big DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,Weiterstadt).

Δ5 acyl lipid desaturase, Pt_des5 Primer 1 (SEQ ID NO: 89)GAG CTC ACA TAA TGG CTC CGG ATG CGG ATA AGC Primer 2 (SEQ ID NO: 90)CTC GAG TTA CGC CCG TCC GGT CAA GGG

The PCR fragment (1428 bp) was cloned into pUC18 with the aid of theSure Clone kit (Pharmacia), the inserted fragment was digested withSacI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

Δ6 acyl lipid desaturase, Pt_des6 Primer 3 (SEQ ID NO: 91)GGA TCC ACA TAA TGG GCA AAG GAG GGG ACG CTC GGG Primer 4 (SEQ ID NO: 92)CTC GAG TTA CAT GGC GGG TCC ATC GGG

The PCR fragment (1451 bp) was cloned into pUC18 with the aid of theSure Clone kit (Pharmacia), the inserted fragment was digested withBamHI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

Δ12 acyl lipid desaturase, Pt_des12 Primer 5 (SEQ ID NO: 93)GGA TCC ACA TAA TGG TTC GCT TTT CAA CAG CC Primer 6 (SEQ ID NO: 94)CTC GAG TTA TTC GCT CGA TAA TTT GC Δ12 acyl lipid desaturase, Pt_des12.2Primer 7 (SEQ ID NO: 95) GGA TCC ACA TAA TGG GTA AGG GAG GTC AAC GPrimer 8 (SEQ ID NO: 96) CTC GAG TCA TGC GGC TTT GTT TCG C

The PCR fragment (1505 bp) was cloned into pUC18 with the aid of theSure Clone kit (Pharmacia), the inserted fragment was digested withBamHI/XhoI, and the fragment was inserted into pYES2 or pYES6 with theaid of suitable restriction cleavage sites.

The plasmid DNA was cleaved with restriction enzyme(s) to match theintroduced cleavage site of the primer sequence, and the fragmentobtained was ligated into the compatible restriction sites of thedephosphorylated yeast/E. coli shuttle vector pYES2 or pYES6, givingrise to pYES derivatives. Following the transformation of E. coli, andDNA minipreparation from the transformants, the orientation of the DNAfragment in the vector was verified by suitable restriction cleavage orsequencing. One clone was used for the DNA maxipreparation with theNucleobond® AX 500 plasmid DNA extraction kit (Macherey-Nagel,Dilringen).

Saccharomyces cerevisiae INVSc1 was transformed with the pYESderivatives and pYES blank vector by means of a PEG/lithium acetateprotocol (Ausubel et al., 1995). Following selection on CMdum agarplates with 2% glucose, pYES derivative transformants and one pYES2transformant were selected for further cultivation and functionalexpression. For pYES6 derivatives, blasticidin was used asantimetabolite. In the case of coexpression based on pYES2 and pYES6,selection was carried out with blasticidin on minimal medium.

Functional Expression of a Desaturase Activity in Yeast Preculture

20 ml of liquid CMdum dropout uracil medium which, however, contains 2%(w/v) of raffinose were inoculated with the transgenic yeast clones(pYES2) and grown for 3 days at 30° C., 200 rpm, until an opticaldensity at 600 nm (OD₆₀₀) of 1.5 to 2 had been reached. If pYES6 wasused as vector, there was additional selection on blasticidin asantimetabolite.

Main Culture

For expression, 20 ml of liquid CMdum dropout uracil medium which,however, contains 2% of raffinose and 1% (v/v) of Tergitol NP-40 weresupplemented with fatty acid substrates to a final concentration of0.003% (w/v). The media were inoculated with the precultures to an OD₆₀₀of 0.05. Expression was induced for 16 hours at an OD₆₀₀ of 0.2, using2% (w/v) of galactose, whereupon the cultures were harvested at an OD₆₀₀of 0.8-1.2.

Fatty Acid Analysis

The total fatty acids were extracted from yeast cultures and analyzed bymeans of gas chromatography. To this end, cells of 5 ml of culture wereharvested by centrifugation (1 000×g, 10 minutes, 4° C.) and washed oncewith 100 mM NaHCO₃, pH 8.0 in order to remove residual medium and fattyacids. To prepare the fatty acid methyl ester(s) (FAMEs or, in thesingular, FAME), the cell sediments were treated for 1 hour at 80° C.with 1 M methanolic H₂SO₄ and 2% (v/v) dimethoxypropane. The FAMEs wereextracted twice with 2 ml of petroleum ether, washed once with 100 mMNaHCO₃, pH 8.0, and once with distilled water, and dried with Na₂SO₄.The organic solvent was evaporated under a stream of argon, and theFAMEs were dissolved in 50 μl of petroleum ether. The samples wereseparated on a ZEBRON-ZB Wax capillary column (30 m, 0.32 mm, 0.25 μm;Phenomenex) in a Hewlett Packard 6850 gas chromatograph equipped withflame ionization detector. The oven temperature was programmed from 70°C. (hold for 1 minute) to 200° C. at a rate of 20° C./minute, then to250° C. (hold for 5 minutes) at a rate of 5° C./minute and finally to260° C. at a rate of 5° C./minute. Nitrogen was used as the carrier gas(4.5 ml/minute at 70° C.). The fatty acids were identified by comparisonwith retention times of FAME standards (SIGMA).

Expression Analysis

The ratios of the fatty acid substrates which had been added and takenup were determined, thus recording the quantity and quality of thedesaturase reaction in accordance with Table 6, Table 7 and Table 8.

The result of the expression of a Phaeodactylum tricornutum Δ6-acyllipid desaturase in yeast:

TABLE 6 pYES2 pYES2-Ptd6 fed with Fatty Acid — — +18:2 +18:3 16:0 13.318.9 28.4 16.7 16:1Δ9 45.4 44.7 12.5 16.9 16:2Δ6, 9 — 4.3 — — 18:0  4.96.3 10.4  9.1 18:1Δ9 36.4 24.1  6.8 11.8 18:2Δ6, 9 — 1.8 — — 18:2Δ9, 12— — 33.4 — 18:3Δ6, 12, 15 — —  4.9 — 18:3Δ9, 12, 15 — — 43.1 18:4Δ6, 9,12, 15 — — —  2.3

The data represent mol % of corresponding cis-fatty acids.

Result of the expression of a Phaeodactylum tricornutum Δ5-acyl lipiddesaturase in yeast:

TABLE 7 Fatty pYES2 pYES_PtD5 construct fed with acid Blank Control 18:218:3 20: 

 Δ8-20:1Δ11 20:2Δ11,14 20:3Ω3 20:3Ω6 16:0Δ 16.9 20.4 27.7 24.4 16.2 2117.6 19.5 22.8 16:1Δ9 44.7 44.1 13.2 9.6 37.4 39.4 38.3 36.9 30.7 18: 

6.1 6.9 10.54 9.8 4.7 7.9 6.3 6.8 8.2 18: 

 Δ9 31.72 28.1 8.77 6 15 26 29.5 25.6 21.1 18: 

 Δ5,9 0.17 0 0 0 0.09 0.21 0.09 9 18: 

 Δ9,12 — 39.7 — — — — — — 18: 

 Δ9,12,15 — 49.9 — — — — — 20: 

 Δ8 — — 25.5 — — — — 20: 

 Δ11 — — — 5.41 — — — 20: 

 Δ5,11 — — — 0.21 — — — 20: 

 Δ11,14 — — — — — 6.48 — — 20: 

 Δ5,11,14 — 0.76 — — 20: 

 Δ11,14,17 — — — — — — 9.83 — 20: 

 Δ8,11,14 — — — — — — — 13.69 20: 

 Δ5,11,14,17 — — — — — — 1.16 — 20: 

 Δ5,8,11,14 — — — — — — — 3.08

The data represent mol % fatty acids of cis-fatty acids.

Further feeding experiments have revealed that C18:1Δ9 was notdesaturated in the presence of C18:2Δ9,11 or C18:3Δ9,12,15 or C20:1Δ8fatty acids, while C18:1 is also desaturated in the presence ofC20:1Δ11, C20:2Δ11,14 and C20:3Δ8,11,14. Also, no desaturation tookplace in the presence of C20:3Δ8,11,14.

When using the protease-deficient yeast strain C13BYS86 (Kunze I. etal., Biochemica et Biophysica Acta (1999) 1410:287-298) for expressingthe Phaeodactylum tricornutum Δ5-desaturase on complete medium withblasticidin, it was found that C20:4 Δ8,11,14,17 as substrate ofΔ5-desaturase gave a conversion rate of 20% and was thus equally wellconverted as C20:3 Δ8,11,14. As an alternative, the auxotrophism markersleu2, ura3 or his can also be used for gene expression.

In a further coexpression experiment of Phaeodactylum 45-desaturase andPhyscomitrella Δ6-elongase, the strain used was UTL7A (Warnecke et al.,J. Biol. Chem. (1999) 274(19):13048-13059), 45-desaturase convertingapproximately 10% of C20:3 Δ8,11,14 into C20:4 Δ5,8,11,14.

Further feeding experiment with a wide range of other fatty acids aloneor in combination (for example linoleic acid, 20:3 Δ5,11,14-fatty acid,α- or γ-linolenic acid, stearidonic acid, arachidonic acid,eicosapentaenoic acid and the like) can be carried out for confirmingthe substrate specificity and substrate selectivity of these desaturasesin greater detail.

TABLE 8 Result of coexpressing a Phaeodactylum tricornutum Δ5-acyl lipiddesaturase and a moss Δ6-elongase in yeast based on the expressionvectors pYES2 and pYES6 pYES2-Elo pYES2-Elo and pYES6-Ptd5 +18:3 +18:4+18:3 +18:4 16:0 15.0 14.8 15.6 15.1 16:1Δ9 27.7 29.2 27.5 29.0 18:0 5.6  6.3  5.7  6.4 18:1Δ9 17.1 30.8 27.4 31.6 18:3Δ6, 9, 12  7.60 — 7.8 — 18:4Δ6, 9, 12, 15 —  6.71 —  6.4 20:3Δ8, 11, 14  15.92 —  13.55 —20:4Δ5, 8, 11, 14 — —  1.31 — 20:4Δ8, 11, 14, 17 — 11.4 —  10.31 20:5Δ5,8, 11, 14, 17 — — —  0.53

The substrate conversions reveal that the Phaeodactylum Δ5-desaturaseand the Physcomitrella patens Δ6-elongase which were used are suitablewith regard to substrate activity and in particular substratespecificity for producing arachidonic acid or eicosapentaenoic acid withthe aid of sequences according to the invention.

The fragmentation patterns and mass spectra of DMOX derivatives ofstandards and the peak fractions of the fatty acids shown in Tables 6, 7and 8 and identified by GC show, in comparison, identical results, thusconfirming the respective position of the double bond beyond simple GCdetection.

Example 17 Purification of the Desired Product from TransformedOrganisms

The recovery of the desired product from plant material or fungi, algae,ciliates, animal cells or the supernatant of the above-describedcultures can be performed by various methods known in the art. If thedesired product is not secreted from the cells, the cells can beharvested from the culture by low-speed centrifugation, the cells can belysed by standard techniques, such as mechanical force or sonication.Organs of plants can be separated mechanically from other tissue orother organs. Following homogenization, the cell debris is removed bycentrifugation, and the supernatant fraction comprising the solubleproteins is retained for further purification of the desired compounds.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and thesupernatant fraction is retained for further purification.

The supernatant fraction from each purification method is subjected tochromatography with a suitable resin, the desired molecule either beingretained on the chromatography resin while many of the impurities of thesample are not, or the impurities being retained by the resin while thesample is not. These chromatography steps can be repeated if necessary,using the same or different chromatography resins. The skilled worker isfamiliar with the selection of suitable chromatography resins and theirmost effective application for a particular molecule to be purified. Thepurified product can be concentrated by filtration or ultrafiltration,and stored at a temperature at which the stability of the product ismaximized.

A wide spectrum of purification methods is known in the art, and theabove purification method is not intended to be limiting. Thesepurification methods are described, for example, in Bailey, J. E., &011is, D. F., Biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986).

The identity and purity of the isolated compounds may be assessed bytechniques which are standard in the art. They include high-performanceliquid chromatography (HPLC), spectroscopic methods, staining methods,thin-layer chromatography, in particular thin-layer chromatography andflame ionization detection (IATROSCAN, Iatron, Tokyo, Japan), NIRS,enzyme assays or microbiological tests. Such analytical methods arereviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140;Malakhova et al. (1996) Biotekhnologiya 11:27-32; and Schmidt et al.(1998) Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia ofIndustrial Chemistry (1996) Vol. A27, VCH Weinheim, pp. 89-90, pp.521-540, pp. 540-547, pp. 559-566, pp. 575-581 and pp. 581-587; Michal,G (1999) Biochemical Pathways: An Atlas of Biochemistry and MolecularBiology, John Wiley and Sons; Fallon, A., et al. (1987) Applications ofHPLC in Biochemistry in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17.

Equivalents

The skilled worker recognizes, or will be able to ascertain, a number ofequivalents of the specific use forms according to the inventiondescribed herein by using no more than routine experiments. Theseequivalents are intended to be encompassed by the claims.

1. A method for the production of fatty acid esters with an increasedcontent of polyunsaturated fatty acids with at least two double bonds,which comprises introducing, into a fatty-acid-ester-producing organism,at least one nucleic acid sequence selected from the group consisting ofa) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 11, b) nucleic acidsequences which, owing to the degeneracy of the genetic code, areobtained by backtranslating the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 12, c)derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:-11, which encodepolypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ IDNO: 6, SEQ ID NO: 10, or SEQ ID NO: 12 and have at least 50% homology atthe amino acid level or which encode polypeptides with the amino acidsequence shown in SEQ ID NO: 4 and have at least about 96% homology atthe amino acid level, without essentially reducing the enzymatic actionof the polypeptides, growing the organism, and isolating the fatty acidesters present in the organism.
 2. A method as claimed in claim 1,wherein the fatty acid esters produced by the method comprisepolyunsaturated C₁₈-, C₂₀- or C₂₂-fatty acid molecules with at least twodouble bonds in the fatty acid ester.
 3. A method as claimed in claim 1,wherein the C₁₈-, C₂₀- or C₂₂-fatty acid molecules are isolated from theorganism in the form of an oil or lipid.
 4. A method as claimed in claim1, wherein a combination of the nucleic acid sequences SEQ ID NO: 1, SEQID NO: 3 and SEQ ID NO: 9 is introduced into thefatty-acid-ester-producing organism.
 5. A method as claimed in claim 1,wherein the organism is a microorganism, an animal or a plant.
 6. Amethod as claimed in claim 1, wherein the organism is a transgenicplant.
 7. A method as claimed in claim 1, wherein the fatty acid esterscontain C₁₈-, C₂₀- or C₂₂-fatty acids with three, four or five doublebonds in the fatty acid ester.
 8. A method as claimed in claim 1,wherein the polyunsaturated fatty acids contained in the fatty acidesters are liberated.
 9. An isolated nucleic acid sequence encoding apolypeptide with desaturase or elongase activity, selected from thegroup consisting of: a) a nucleic acid sequence with the sequence shownin SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:11, b) nucleic acid sequences which, owing to the degeneracy of thegenetic code, are obtained by backtranslating the amino acid sequencesshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, or SEQID NO: 12, c) derivatives of the nucleic acid sequences shown in SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 11, whichencode polypeptides with the amino acid sequences shown in SEQ ID NO: 2,SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 12 and have at least 50%homology at the amino acid level or which encode polypeptides with theamino acid sequence shown in SEQ ID NO: 4 and have at least about 96%homology at the amino acid level, without essentially reducing theenzymatic action of the polypeptides.
 10. An amino acid sequence encodedby a nucleic acid sequence as claimed in claim
 9. 11. An amino acidsequence as claimed in claim 10 encoded by the sequence shown in SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:
 11. 12. Anucleic acid construct comprising a nucleic acid sequence as claimed inclaim 9, wherein the nucleic acid sequence is linked to one or moreregulatory signals.
 13. A nucleic acid construct as claimed in claim 12,wherein additional biosynthesis genes of the fatty acid or lipidmetabolism are present in the nucleic acid construct.
 14. A nucleic acidconstruct as claimed in claim 13, wherein the biosynthesis gene of thefatty acid or lipid metabolism which is present in the nucleic acidconstruct is a gene selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyl transferase(s), fatty acidsynthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases,allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s).15. A vector comprising a nucleic acid sequence as claimed in claim 9 ora nucleic acid construct comprising said nucleic acid sequence.
 16. Anorganism comprising the isolated nucleic acid sequence of claim 9, aconstruct comprising the nucleic acid sequence linked to one or moreregulatory signals, or a vector comprising the nucleic acid sequence orthe construct, wherein the organism is a microorganism, a yeast, or aplant or plant cell.
 17. A transgenic plant or plant cell comprising theisolated nucleic acid sequence of claim 9 or a construct comprising thenucleic acid sequence linked to one or more regulatory signals.
 18. Theuse of a nucleic acid sequence as claimed in claim 9 or of a nucleicacid construct comprising said nucleic acid sequence for generatingtransgenic plants.
 19. An antisense nucleic acid molecule comprising thecomplementary sequence of the nucleic acid as claimed in claim
 9. 20.The use of an oil, lipid or fatty acid, or a fraction thereof, obtainedfrom the method of claim 1 in feed, foodstuffs, cosmetics orpharmaceuticals.